The pathogenetic significance of exhausted T cells in a mouse model of mature B cell neoplasms.

Dysfunctional anti-tumor immunity has been implicated in the pathogenesis of mature B cell neoplasms, such as multiple myeloma and B cell lymphoma; however, the impact of exhausted T cells on disease development remains unclear. Therefore, the present study investigated the features and pathogenetic significance of exhausted T cells using a mouse model of de novo mature B cell neoplasms, which is likely to show immune escape similar to human patients. The results revealed a significant increase in PD-1+ Tim-3- and PD-1+ Tim-3+ T cells in sick mice. Furthermore, PD-1+ Tim-3+ T cells exhibited direct cytotoxicity with a short lifespan, showing transcriptional similarities to terminally exhausted T cells. On the other hand, PD-1+ Tim-3- T cells not only exhibited immunological responsiveness but also retained stem-like transcriptional features, suggesting that they play a role in the long-term maintenance of anti-tumor immunity. In PD-1+ Tim-3- and PD-1+ Tim-3+ T cells, the transcription factors Tox and Nr4a2, which reportedly contribute to the progression of T cell exhaustion, were up-regulated in vivo. These transcription factors were down-regulated by IMiDs in our in vitro T cell exhaustion analyses. The prevention of excessive T cell exhaustion may maintain effective anti-tumor immunity to cure mature B cell neoplasms.

YAP1/TAZ activity maintains vascular integrity and organismal survival.

Radiation therapy is one of the major treatment modalities for patients with cancers. However, ionizing radiation (IR) damages not only cancer cells but also the surrounding vascular endothelial cells (ECs). Hippo pathway effector genes Yap1 and Taz are the two transcriptional coactivators that have crucial roles in tissue homeostasis and vascular integrity in various organs. However, their function in adult ECs at the steady state and after IR is poorly understood. Here, we report sex- and context-dependent roles of endothelial YAP1/TAZ in maintaining vascular integrity and organismal survival. EC-specific Yap1/Taz deletion compromised systemic vascular integrity, resulting in lethal circulation failure preferentially in male mice. Furthermore, EC-specific Yap1/Taz deletion induced acute lethality upon sublethal IR that was closely associated with exacerbated systemic vascular dysfunction and circulation failure. Consistent with these findings, RNA-seq analysis revealed downregulation of tight junction genes in Yap1/Taz-deleted ECs. Collectively, our findings highlight the importance of endothelial YAP1/TAZ for maintaining adult vascular function, which may provide clinical implications for preventing organ injury after radiation therapy.

Bmi1 counteracts hematopoietic stem cell aging by repressing target genes and enforcing the stem cell gene signature.

Polycomb-group proteins are critical regulators of stem cells. We previously demonstrated that Bmi1, a component of polycomb repressive complex 1, defines the regenerative capacity of hematopoietic stem cells (HSCs). Here, we attempted to ameliorate the age-related decline in HSC function by modulating Bmi1 expression. The forced expression of Bmi1 did not attenuate myeloid-biased differentiation of aged HSCs. However, single cell transplantation assays revealed that the sustained expression of Bmi1 augmented the multi-lineage repopulating capacity of aged HSCs. Chromatin immunoprecipitation-sequencing of Bmi1 combined with an RNA sequence analysis showed that the majority of Bmi1 direct target genes are developmental regulator genes marked with a bivalent histone domain. The sustained expression of Bmi1 strictly maintained the transcriptional repression of their target genes and enforced expression of HSC signature genes in aged HSCs. Therefore, the manipulation of Bmi1 expression is a potential approach against impairments in HSC function with aging.

Bcor insufficiency promotes initiation and progression of myelodysplastic syndrome.

BCOR, encoding BCL-6 corepressor (BCOR), is X-linked and targeted by somatic mutations in various hematological malignancies including myelodysplastic syndrome (MDS). We previously reported that mice lacking Bcor exon 4 (Bcor ΔE4/y ) in the hematopoietic compartment developed NOTCH-dependent acute T-cell lymphoblastic leukemia (T-ALL). Here, we analyzed mice lacking Bcor exons 9 and 10 (Bcor ΔE9-10/y ), which express a carboxyl-terminal truncated BCOR that fails to interact with core effector components of polycomb repressive complex 1.1. Bcor ΔE9-10/y mice developed lethal T-ALL in a similar manner to Bcor ΔE4/y mice, whereas Bcor ΔE9-10/y hematopoietic cells showed a growth advantage in the myeloid compartment that was further enhanced by the concurrent deletion of Tet2 Tet2 Δ/Δ Bcor ΔE9-10/y mice developed lethal MDS with progressive anemia and leukocytopenia, inefficient hematopoiesis, and the morphological dysplasia of blood cells. Tet2 Δ/Δ Bcor ΔE9-10/y MDS cells reproduced MDS or evolved into lethal MDS/myeloproliferative neoplasms in secondary recipients. Transcriptional profiling revealed the derepression of myeloid regulator genes of the Cebp family and Hoxa cluster genes in Bcor ΔE9-10/y progenitor cells and the activation of p53 target genes specifically in MDS erythroblasts where massive apoptosis occurred. Our results reveal a tumor suppressor function of BCOR in myeloid malignancies and highlight the impact of Bcor insufficiency on the initiation and progression of MDS.

Forkhead box protein A1 confers resistance to transforming growth factor-ß-induced apoptosis in breast cancer cells through inhibition of Smad3 nuclear translocation.

Transforming growth factor-ß (TGF-ß) induces apoptosis of normal epithelial cells, such as mammary epithelium. Although breast cancer progression associates with acquisition of resistance to TGF-ß-induced apoptosis, the molecular mechanisms underlying this resistance are largely unknown. Here, we show that forkhead box protein A1 (FOXA1), which is known as a pioneer transcription factor, suppresses TGF-ß-induced apoptosis of estrogen receptor-positive breast cancer cells. FOXA1 is found to inhibit nuclear translocation of Smad3, a key transcription factor downstream of TGF-ß signaling, through suppression of the binding of Smad3 to the nuclear import receptor importin7. Furthermore, RNA sequencing analyses show that knockdown of FOXA1 upregulates Smad3-mediated proapoptotic gene expression. These results demonstrate that FOXA1 as a potent survival factor that suppresses TGF-ß-induced apoptosis by inhibiting Smad3 signaling in estrogen receptor-positive breast cancer cells. Thus, we provide evidence for the first time that FOXA1 localizing to the cytoplasm negatively regulates Smad3-induced apoptosis in TGF-ß-mediated signal transduction.

Role for tyrosine phosphorylation of SUV39H1 histone methyltransferase in enhanced trimethylation of histone H3K9 via neuregulin-1/ErbB4 nuclear signaling.

Protein-tyrosine kinases transmit signals by phosphorylating their substrates in diverse cellular events. The receptor-type tyrosine kinase ErbB4, a member of the epidermal growth factor receptor subfamily, is activated and proteolytically cleaved upon ligand stimulation, and the cleaved ErbB4 intracellular domain (4ICD) is released into the cytoplasm and the nucleus. We previously showed that generation of nuclear 4ICD by neuregulin-1 (NRG-1) stimulation enhances the levels of trimethylation of histone H3 at lysine 9 (H3K9me3). However, it remains unclear how nuclear 4ICD enhances H3K9me3 levels. Here we show that the histone H3K9 methyltransferase SUV39H1 associates with NRG-1/ErbB4-mediated H3K9me3. Knockdown of SUV39H1 blocked NRG-1-mediated enhancement of the levels of H3K9me3. Nuclear 4ICD was found to phosphorylate SUV39H1 primarily at Tyr-297, -303, and -308 that are conserved among humans, mice, and flies. Furthermore, knockdown-rescue experiments showed that the unphosphorylatable SUV39H1 mutant (3 YF) was incapable of enhancing the levels of H3K9me3 upon NRG-1 stimulation. These results suggest that nuclear ErbB4 enhances H3K9me3 levels through tyrosine phosphorylation of SUV39H1 in NRG-1/ErbB4 signal-mediated chromatin remodeling.

Setdb1 maintains hematopoietic stem and progenitor cells by restricting the ectopic activation of nonhematopoietic genes.

Setdb1, also known as Eset, is a methyltransferase that catalyzes trimethylation of H3K9 (H3K9me3) and plays an essential role in the silencing of endogenous retroviral elements (ERVs) in the developing embryo and embryonic stem cells (ESCs). Its role in somatic stem cells, however, remains unclear because of the early death of Setdb1-deficient embryos. We demonstrate here that Setdb1 is the first H3K9 methyltransferase shown to be essential for the maintenance of hematopoietic stem and progenitor cells (HSPCs) in mice. The deletion of Setdb1 caused the rapid depletion of hematopoietic stem and progenitor cells (HSPCs), as well as leukemic stem cells. In contrast to ESCs, ERVs were largely repressed in Setdb1-deficient HSPCs. A list of nonhematopoietic genes was instead ectopically activated in HSPCs after reductions in H3K9me3 levels, including key gluconeogenic enzyme genes fructose-1,6-bisphosphatase 1 (Fbp1) and Fbp2 The ectopic activation of gluconeogenic enzymes antagonized glycolysis and impaired ATP production, resulting in a compromised repopulating capacity of HSPCs. Our results demonstrate that Setdb1 maintains HSPCs by restricting the ectopic activation of nonhematopoietic genes detrimental to their function and uncover that the gluconeogenic pathway is one of the critical targets of Setdb1 in HSPCs.

Enhancement of TGF-ß-induced Smad3 activity by c-Abl-mediated tyrosine phosphorylation of its coactivator SKI-interacting protein (SKIP).

c-Abl is a non-receptor-type tyrosine kinase that plays an important role in cell proliferation, migration, apoptosis, and fibrosis. Furthermore, although c-Abl is involved in transforming growth factor-ß (TGF-ß) signaling, its molecular functions in TGF-ß signaling are not fully understood. Here, we found that c-Abl phosphorylates SKI-interacting protein (SKIP), a nuclear cofactor of the transcription factor Smad3. The c-Abl inhibitor imatinib suppressed TGF-ß-induced expression of Smad3 targets as well as SKIP/Smad3 interaction. TGF-ß-stimulation induced tyrosine phosphorylation of SKIP, and this phosphorylation was suppressed by imatinib. Tyr292, Tyr430, and Tyr433 residues in SKIP were shown to be involved in c-Abl-mediated phosphorylation. Phosphomimetic glutamic acid substitution at Tyr292 in SKIP enhanced, whereas its phospho-dead phenylalanine substitution attenuated TGF-ß-induced SKIP/Smad3 interaction. Moreover, the phosphomimetic mutant of SKIP augmented transcriptional activity of Smad3. Taken together, these results suggest that c-Abl phosphorylates SKIP mainly at Tyr292 and promotes SKIP/Smad3 interaction for the full activation of TGF-ß/Smad3 signaling.

Ezh2 loss in hematopoietic stem cells predisposes mice to develop heterogeneous malignancies in an Ezh1-dependent manner.

Recent genome sequencing revealed inactivating mutations in EZH2, which encodes an enzymatic component of polycomb-repressive complex 2 (PRC2), in patients with myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPNs), and MDS/MPN overlap disorders. We herein demonstrated that the hematopoietic-specific deletion of Ezh2 in mice induced heterogeneous hematopoietic malignancies. Myelodysplasia was detected in mice following the deletion of Ezh2, and resulted in the development of MDS and MDS/MPN. Thrombocytosis was induced by Ezh2 loss and sustained in some mice with myelodysplasia. Although less frequent, Ezh2 loss also induced T-cell acute lymphoblastic leukemia and the clonal expansion of B-1a B cells. Gene expression profiling showed that PRC2 target genes were derepressed upon the deletion of Ezh2 in hematopoietic stem and progenitor cells, but were largely repressed during the development of MDS and MDS/MPN. Chromatin immunoprecipitation-sequence analysis of trimethylation of histone H3 at lysine 27 (H3K27me3) revealed a compensatory function of Ezh1, another enzymatic component of PRC2, in this process. The deletion of Ezh1 alone did not cause dysplasia or any hematologic malignancies in mice, but abolished the repopulating capacity of hematopoietic stem cells when combined with Ezh2 loss. These results clearly demonstrated an essential role of Ezh1 in the pathogenesis of hematopoietic malignancies induced by Ezh2 insufficiency, and highlighted the differential functions of Ezh1 and Ezh2 in hematopoiesis.

Loss of TET2 has dual roles in murine myeloproliferative neoplasms: disease sustainer and disease accelerator.

Acquired mutations of JAK2 and TET2 are frequent in myeloproliferative neoplasms (MPNs). We examined the individual and cooperative effects of these mutations on MPN development. Recipients of JAK2V617F cells developed primary myelofibrosis-like features; the addition of loss of TET2 worsened this JAK2V617F-induced disease, causing prolonged leukocytosis, splenomegaly, extramedullary hematopoiesis, and modestly shorter survival. Double-mutant (JAK2V617F plus loss of TET2) myeloid cells were more likely to be in a proliferative state than JAK2V617F single-mutant myeloid cells. In a serial competitive transplantation assay, JAK2V617F cells resulted in decreased chimerism in the second recipients, which did not develop MPNs. In marked contrast, cooperation between JAK2V617F and loss of TET2 developed and maintained MPNs in the second recipients by compensating for impaired hematopoietic stem cell (HSC) functioning. In-vitro sequential colony formation assays also supported the observation that JAK2V617F did not maintain HSC functioning over the long-term, but concurrent loss of TET2 mutation restored it. Transcriptional profiling revealed that loss of TET2 affected the expression of many HSC signature genes. We conclude that loss of TET2 has two different roles in MPNs: disease accelerator and disease initiator and sustainer in combination with JAK2V617F.

Role for Tyrosine Phosphorylation of A-kinase Anchoring Protein 8 (AKAP8) in Its Dissociation from Chromatin and the Nuclear Matrix.

Protein-tyrosine phosphorylation regulates a wide variety of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine kinases induce global nuclear structure changes, which we called chromatin structural changes. However, the mechanisms are not fully understood. In this study we identify protein kinase A anchoring protein 8 (AKAP8/AKAP95), which associates with chromatin and the nuclear matrix, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of AKAP8 is induced by several tyrosine kinases, such as Src, Fyn, and c-Abl but not Syk. Nucleus-targeted Lyn and c-Src strongly dissociate AKAP8 from chromatin and the nuclear matrix in a kinase activity-dependent manner. The levels of tyrosine phosphorylation of AKAP8 are decreased by substitution of multiple tyrosine residues on AKAP8 into phenylalanine. Importantly, the phenylalanine mutations of AKAP8 inhibit its dissociation from nuclear structures, suggesting that the association/dissociation of AKAP8 with/from nuclear structures is regulated by its tyrosine phosphorylation. Furthermore, the phenylalanine mutations of AKAP8 suppress the levels of nuclear tyrosine kinase-induced chromatin structural changes. In contrast, AKAP8 knockdown increases the levels of chromatin structural changes. Intriguingly, stimulation with hydrogen peroxide induces chromatin structural changes accompanied by the dissociation of AKAP8 from nuclear structures. These results suggest that AKAP8 is involved in the regulation of chromatin structural changes through nuclear tyrosine phosphorylation.

c-Abl-mediated tyrosine phosphorylation of JunB is required for Adriamycin-induced expression of p21.

The non-receptor-type tyrosine kinase c-Abl functions as a cytoplasmic signal transducer upon activation of cell-surface receptors. c-Abl is also involved in DDR (DNA-damage response), which is initiated in the nucleus, whereas its molecular functions in DDR are not fully understood. In the present study, we found that c-Abl phosphorylates JunB, a member of the AP-1 (activator protein 1) transcription factor family. Because JunB was suggested to be involved in DDR, we analysed the role of c-Abl-mediated phosphorylation of JunB in DDR. We first analysed phosphorylation sites of JunB and found that c-Abl majorly phosphorylates JunB at Tyr(173), Tyr(182) and Tyr(188). Because c-Abl promotes expression of the cyclin-dependent kinase inhibitor p21 upon stimulation with the DNA-damaging agent Adriamycin (doxorubicin), we analysed the involvement of JunB in Adriamycin-induced p21 expression. We found that JunB suppresses p21 induction through inhibition of its promoter activity. The phosphomimetic JunB, which was generated by glutamic acid substitutions at the phosphorylation sites, failed to repress p21 induction. Recruitment of JunB to the p21 promoter was promoted by Adriamycin stimulation and was further enhanced by co-treatment with the c-Abl inhibitor imatinib. The phosphomimetic glutamic acid substitutions in JunB or Adriamycin treatment impaired the JunB-c-Fos transcription factor complex formation. Taken together, these results suggest that, although JunB represses p21 promoter activity, c-Abl phosphorylates JunB and conversely inhibits its suppressive role on p21 promoter activity upon Adriamycin stimulation. Therefore JunB is likely to be a key target of c-Abl in expression of p21 in Adriamycin-induced DDR.

Design, Synthesis, and Biological Evaluation of Beauveriolide Analogues Bearing Photoreactive Amino Acids.

Beauveriolides I and III, which are naturally occurring cyclodepsipeptides, have been reported to bind to sterol O-acyltransferase (SOAT), inhibiting its ability to synthesize cholesteryl esters. To facilitate an analysis of the binding site(s) of these compounds, we designed beauveriolide analogues 1a-d wherein the Leu or D-allo-Ile residue was replaced by photoreactive amino acids possessing methyldiazirine or trifluoromethyldiazirine in the side chains. The methyldiazirine moiety was installed by reaction of methyl ketones with liquid ammonia to provide imine intermediates, followed by treatment with hydroxylamine-O-sulfonic acid to provide the diaziridines. Subsequent oxidation gave methyldiazirines. In contrast, trifluoromethyldiazirine derivatives were prepared from trifluoromethyl ketones via the oxime intermediates, which were transformed into diaziridines. Subsequent oxidation afforded trifluoromethyldiazirines. The synthesized photoreactive amino acids 3a-d were coupled with 3-hydroxy-4-methyloctanoic acid 4 and dipeptide 5, followed by macrolactamization to provide beauveriolide analogues 1a-d. The SOAT inhibitory activities of 1a-d were found to be as potent as those of beauveriolides I and III. Moreover, 1a-d inhibited SOAT1 selectively rather than SOAT2, which was also consistent with the behavior of beauveriolides I and III.

Activation of the prereplication complex is blocked by mimosine through reactive oxygen species-activated ataxia telangiectasia mutated (ATM) protein without DNA damage.

Mimosine is an effective cell synchronization reagent used for arresting cells in late G1 phase. However, the mechanism underlying mimosine-induced G1 cell cycle arrest remains unclear. Using highly synchronous cell populations, we show here that mimosine blocks S phase entry through ATM activation. HeLa S3 cells are exposed to thymidine for 15 h, released for 9 h by washing out the thymidine, and subsequently treated with 1 mM mimosine for a further 15 h (thymidine → mimosine). In contrast to thymidine-induced S phase arrest, mimosine treatment synchronizes >90% of cells at the G1-S phase boundary by inhibiting the transition of the prereplication complex to the preinitiation complex. Mimosine treatment activates ataxia telangiectasia mutated (ATM)/ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint signaling without inducing DNA damage. Inhibition of ATM activity is found to induce mimosine-arrested cells to enter S phase. In addition, ATM activation by mimosine treatment is mediated by reactive oxygen species (ROS). These results suggest that, upon mimosine treatment, ATM blocks S phase entry in response to ROS, which prevents replication fork stalling-induced DNA damage.

Overexpression of zinc-finger protein 777 (ZNF777) inhibits proliferation at low cell density through down-regulation of FAM129A.

Krüppel-associated box-containing zinc finger proteins (KRAB-ZFPs) regulate a wide range of cellular processes. KRAB-ZFPs have a KRAB domain, which binds to transcriptional corepressors, and a zinc finger domain, which binds to DNA to activate or repress gene transcription. Here, we characterize ZNF777, a member of KRAB-ZFPs. We show that ZNF777 localizes to the nucleus and inducible overexpression of ZNF777 inhibits cell proliferation in a manner dependent on its zinc finger domain but independent of its KRAB domain. Intriguingly, ZNF777 overexpression drastically inhibits cell proliferation at low cell density but slightly inhibits cell proliferation at high cell density. Furthermore, ZNF777 overexpression decreases the mRNA level of FAM129A irrespective of cell density. Importantly, the protein level of FAM129A strongly decreases at low cell density, but at high cell density the protein level of FAM129A does not decrease to that observed at low cell density. ZNF777-mediated inhibition of cell proliferation is attenuated by overexpression of FAM129A at low cell density. Furthermore, ZNF777-mediated down-regulation of FAM129A induces moderate levels of the cyclin-dependent kinase inhibitor p21. These results suggest that ZNF777 overexpression inhibits cell proliferation at low cell density and that p21 induction by ZNF777-mediated down-regulation of FAM129A plays a role in inhibition of cell proliferation.

Nuclear ErbB4 signaling through H3K9me3 is antagonized by EGFR-activated c-Src.

The ErbB family of receptor tyrosine kinases comprises four members: epidermal growth factor receptor (EGFR)/ErbB1, HER2/ErbB2, ErbB3 and ErbB4, and plays roles in signal transduction at the plasma membrane upon ligand stimulation. Stimulation with neuregulin-1 (NRG-1) cleaves ErbB4 and releases the ErbB4 intracellular domain (4ICD) that translocates into the nucleus to control gene expression. However, little is known about the regulation of 4ICD nuclear signaling through tyrosine phosphorylation. We show here that 4ICD nuclear signaling is antagonized by EGF-induced c-Src activation through EGFR. Generation of 4ICD by NRG-1 leads to increased levels of trimethylated histone H3 on lysine 9 (H3K9me3) in a manner dependent on the nuclear accumulation of 4ICD and its tyrosine kinase activity. Once EGF activates c-Src downstream of EGFR concomitantly with NRG-1-induced ErbB4 activation, c-Src associates with phospho-Tyr950 and phospho-Tyr1056 on 4ICD, thereby decreasing nuclear accumulation of 4ICD and inhibiting an increase of H3K9me3 levels. Moreover, 4ICD-induced transcriptional repression of the human telomerase reverse transcriptase (hTERT) is inhibited by EGF-EGFR-Src signaling. Thus, our findings reveal c-Src-mediated inhibitory regulation of ErbB4 nuclear signaling upon EGFR activation.

c-Abl induces stabilization of histone deacetylase 1 (HDAC1) in a kinase activity-dependent manner.

c-Abl is a non-receptor-type tyrosine kinase that regulates various cellular events, including cell proliferation, differentiation, and apoptosis, through phosphorylation of cytoplasmic and nuclear targets. Although we showed that c-Abl induces histone deacetylation, the molecular mechanisms of this phenomenon are largely unknown. Here, we analyzed the effect of c-Abl on the expression of histone deacetylase 1 (HDAC1), because c-Abl was shown to be involved in maintenance of nuclear protein levels of HDAC1. Co-transfection of HDAC1 with c-Abl increased the levels of HDAC1 protein in a kinase activity-dependent manner without affecting its mRNA levels. Treatment with the proteasome inhibitor MG132 increased protein levels of HDAC1 in cells transfected with HDAC1 but not in cells co-transfected with HDAC1 and c-Abl. Among class I HDACs, knockdown of endogenous c-Abl preferentially suppressed endogenous protein levels of HDAC1, suggesting that c-Abl stabilizes HDAC1 protein by inhibiting its proteasomal degradation. Subcellular fractionation showed that the stabilization of HDAC1 by c-Abl occurred in the nucleus. Despite the fact that HDAC1 was phosphorylated by co-expression with c-Abl, stabilization of HDAC1 by c-Abl was not affected by mutations in its sites phosphorylated by c-Abl. Co-expression with HDAC1 and nuclear-targeted c-Abl did not affect HDAC1 stabilization. Therefore, these results suggest that c-Abl induces HDAC1 stabilization possibly through phosphorylation of a cytoplasmic target that is involved in proteasomal degradation of HDAC1.

Phosphorylation of KRAB-associated protein 1 (KAP1) at Tyr-449, Tyr-458, and Tyr-517 by nuclear tyrosine kinases inhibits the association of KAP1 and heterochromatin protein 1α (HP1α) with heterochromatin.

Protein tyrosine phosphorylation regulates a wide range of cellular processes at the plasma membrane. Recently, we showed that nuclear tyrosine phosphorylation by Src family kinases (SFKs) induces chromatin structural changes. In this study, we identify KRAB-associated protein 1 (KAP1/TIF1ß/TRIM28), a component of heterochromatin, as a nuclear tyrosine-phosphorylated protein. Tyrosine phosphorylation of KAP1 is induced by several tyrosine kinases, such as Src, Lyn, Abl, and Brk. Among SFKs, Src strongly induces tyrosine phosphorylation of KAP1. Nucleus-targeted Lyn potentiates tyrosine phosphorylation of KAP1 compared with intact Lyn, but neither intact Fyn nor nucleus-targeted Fyn phosphorylates KAP1. Substitution of the three tyrosine residues Tyr-449/Tyr-458/Tyr-517, located close to the HP1 binding-motif, into phenylalanine ablates tyrosine phosphorylation of KAP1. Immunostaining and chromatin fractionation show that Src and Lyn decrease the association of KAP1 with heterochromatin in a kinase activity-dependent manner. KAP1 knockdown impairs the association of HP1α with heterochromatin, because HP1α associates with KAP1 in heterochromatin. Intriguingly, tyrosine phosphorylation of KAP1 decreases the association of HP1α with heterochromatin, which is inhibited by replacement of endogenous KAP1 with its phenylalanine mutant (KAP1-Y449F/Y458F/Y517F, KAP1-3YF). In DNA damage, KAP1-3YF repressed transcription of p21. These results suggest that nucleus-localized tyrosine kinases, including SFKs, phosphorylate KAP1 at Tyr-449/Tyr-458/Tyr-517 and inhibit the association of KAP1 and HP1α with heterochromatin.

Formation of long and winding nuclear F-actin bundles by nuclear c-Abl tyrosine kinase.

The non-receptor-type tyrosine kinase c-Abl is involved in actin dynamics in the cytoplasm. Having three nuclear localization signals (NLSs) and one nuclear export signal, c-Abl shuttles between the nucleus and the cytoplasm. Although monomeric actin and filamentous actin (F-actin) are present in the nucleus, little is known about the relationship between c-Abl and nuclear actin dynamics. Here, we show that nuclear-localized c-Abl induces nuclear F-actin formation. Adriamycin-induced DNA damage together with leptomycin B treatment accumulates c-Abl into the nucleus and increases the levels of nuclear F-actin. Treatment of c-Abl-knockdown cells with Adriamycin and leptomycin B barely increases the nuclear F-actin levels. Expression of nuclear-targeted c-Abl (NLS-c-Abl) increases the levels of nuclear F-actin even without Adriamycin, and the increased levels of nuclear F-actin are not inhibited by inactivation of Abl kinase activity. Intriguingly, expression of NLS-c-Abl induces the formation of long and winding bundles of F-actin within the nucleus in a c-Abl kinase activity-dependent manner. Furthermore, NLS-c-AblΔC, which lacks the actin-binding domain but has the full tyrosine kinase activity, is incapable of forming nuclear F-actin and in particular long and winding nuclear F-actin bundles. These results suggest that nuclear c-Abl plays critical roles in actin dynamics within the nucleus.

Polycomb repressive complex 1.1 coordinates homeostatic and emergency myelopoiesis.

Polycomb repressive complex (PRC) 1 regulates stem cell fate by mediating mono-ubiquitination of histone H2A at lysine 119. While canonical PRC1 is critical for hematopoietic stem and progenitor cell (HSPC) maintenance, the role of non-canonical PRC1 in hematopoiesis remains elusive. PRC1.1, a non-canonical PRC1, consists of PCGF1, RING1B, KDM2B, and BCOR. We recently showed that PRC1.1 insufficiency induced by the loss of PCGF1 or BCOR causes myeloid-biased hematopoiesis and promotes transformation of hematopoietic cells in mice. Here we show that PRC1.1 serves as an epigenetic switch that coordinates homeostatic and emergency hematopoiesis. PRC1.1 maintains balanced output of steady-state hematopoiesis by restricting C/EBPα-dependent precocious myeloid differentiation of HSPCs and the HOXA9- and ß-catenin-driven self-renewing network in myeloid progenitors. Upon regeneration, PRC1.1 is transiently inhibited to facilitate formation of granulocyte-macrophage progenitor (GMP) clusters, thereby promoting emergency myelopoiesis. Moreover, constitutive inactivation of PRC1.1 results in unchecked expansion of GMPs and eventual transformation. Collectively, our results define PRC1.1 as a novel critical regulator of emergency myelopoiesis, dysregulation of which leads to myeloid transformation.