N-terminal Transcriptional Activation Research Articles

E242-E261 region of MYC regulates liquid-liquid phase separation and tumor growth by providing negative charges

MYC is one of the most extensively studied oncogenic proteins and is closely associated with the occurrence and progression of many tumors. Previous studies have shown that MYC regulates cell fate through its liquid-liquid phase separation (LLPS) mechanism, which is dependent on two disordered domains within its N-terminal transcriptional activation regions. In this study, we revealed that the negatively charged conserved region (E242-E261) of the MYC protein controls its condensation formation and irreversible aggregation through multivalent electrostatic interactions (MEIs). Furthermore, deletion or mutation of the E242-E261 amino acids in the MYC protein enhances the transcriptional function of MYC by altering its aggregation capacity and subsequently promoting cancer cell proliferation. The discovery of the negatively charged region and its regulatory action on the phase separation of MYC provides a new understanding on the aggregation and function of MYC.

Substitutions at non-conserved, rheostat positions in intrinsically disordered regions of CIITA modulate its function.

Personalized medicine requires understanding which exome changes cause medically relevant phenotypes. To predict exome phenotypes, existing algorithms rely on textbook rules for amino acid substitutions that were derived from decades of studies biased towards conserved positions. Recent studies targeting non-conserved positions found a special class of “rheostat” positions. Substitutions at these positions exhibited a range of functional outcomes that were not predicted by existing rules. Understanding such rheostat positions is critical to improve predictions. To that end, we tested rheostat positions in globular proteins and found substitutions altered protein dynamics. Here, we extended our studies to the extremely dynamic and non-conserved intrinsically disordered (ID) proteins using the Class II transactivator (CIITA) as our model. CIITA is the master transcriptional activator of the MHC class II genes. Sequence analyses and experiments show that the N-terminal transcriptional activation domain of CIITA has positions with high and low probabilities for ID. This region has putative binding for interaction with multivalent partners. We hypothesized that rheostats would be in positions with low ID probabilities. We measured the effects of 82 single substitutions across 7 positions on (i) expression, (ii) transcriptional activation, and (iii) overall biological output. We observed modest effects on expression, identified strong rheostats for transcriptional activation and overall biological output. Surprisingly, rheostats were found in both regions of low and high ID probabilities. Furthermore, substitutions at just 7 of the 1130 positions sampled nearly the entire range of possible transcription activation levels. These results show that this multi-valent, highly non-conserved, ID protein has a substitution sensitivity that is similar to globular soluble and integral membrane proteins. Finally, retrospective inspection of alanine scans of other transcription activation domains suggest that strong rheostat positions exist in other many other IDPs.

Functional interaction between ELL transcription elongation factor and Epe1 reveals the role of Epe1 in the regulation of transcription outside heterochromatin.

Eleven-nineteen lysine-rich leukemia (ELL) is a eukaryotic RNA polymerase II transcription elongation factor. In Schizosaccharomyces pombe, it is important for survival under genotoxic stress conditions. However, the molecular basis underlying this function of ELL in S. pombe is yet to be deciphered. Here, we carried out a genetic screen to identify multicopy suppressor(s) that could restore normal growth of ell1 deletion mutant in the presence of DNA damaging agent. Sequence analysis of the identified suppressors revealed the anti-silencing protein, Epe1, as one of the suppressors of ell1 deletion associated genotoxic stress sensitivity. Our results further demonstrate that the overexpression of Epe1 could suppress all other phenotypes associated with the absence of Ell1. Moreover, transcriptional defect of ell1Δ strain could also be alleviated by the overexpression of Epe1. Epe1 also showed a physical interaction with Ell1. Interestingly, we also observed that the region of Epe1 encompassing 403-948 amino acids was indispensable for all the above functions. Furthermore, our results show that the overexpression of Epe1 causes increased H3K9 acetylation and RNA polymerase II recruitment. Taken together, our results show a functional interaction between Epe1 and Ell1, and this function is independent of the well-known JmjC and N-terminal transcriptional activation domains of Epe1 in S. pombe.

Regulation of ectopic heterochromatin-mediated epigenetic diversification by the JmjC family protein Epe1.

H3K9 methylation (H3K9me) is a conserved marker of heterochromatin, a transcriptionally silent chromatin structure. Knowledge of the mechanisms for regulating heterochromatin distribution is limited. The fission yeast JmjC domain-containing protein Epe1 localizes to heterochromatin mainly through its interaction with Swi6, a homologue of heterochromatin protein 1 (HP1), and directs JmjC-mediated H3K9me demethylation in vivo. Here, we found that loss of epe1 (epe1Δ) induced a red-white variegated phenotype in a red-pigment accumulation background that generated uniform red colonies. Analysis of isolated red and white colonies revealed that silencing of genes involved in pigment accumulation by stochastic ectopic heterochromatin formation led to white colony formation. In addition, genome-wide analysis of red- and white-isolated clones revealed that epe1Δ resulted in a heterogeneous heterochromatin distribution among clones. We found that Epe1 had an N-terminal domain distinct from its JmjC domain, which activated transcription in both fission and budding yeasts. The N-terminal transcriptional activation (NTA) domain was involved in suppression of ectopic heterochromatin-mediated red-white variegation. We introduced a single copy of Epe1 into epe1Δ clones harboring ectopic heterochromatin, and found that Epe1 could reduce H3K9me from ectopic heterochromatin but some of the heterochromatin persisted. This persistence was due to a latent H3K9me source embedded in ectopic heterochromatin. Epe1H297A, a canonical JmjC mutant, suppressed red-white variegation, but entirely failed to remove already-established ectopic heterochromatin, suggesting that Epe1 prevented stochastic de novo deposition of ectopic H3K9me in an NTA-dependent but JmjC-independent manner, while its JmjC domain mediated removal of H3K9me from established ectopic heterochromatin. Our results suggest that Epe1 not only limits the distribution of heterochromatin but also controls the balance between suppression and retention of heterochromatin-mediated epigenetic diversification.

Conserved RNA binding activity of a Yin-Yang 1 homologue in the ova of the purple sea urchin Strongylocentrotus purpuratus

Yin-Yang 1 (YY1) is a highly conserved transcription factor possessing RNA-binding activity. A putative YY1 homologue was previously identified in the developmental model organism Strongylocentrotus purpuratus (the purple sea urchin) by genomic sequencing. We identified a high degree of sequence similarity with YY1 homologues of vertebrate origin which shared 100% protein sequence identity over the DNA- and RNA-binding zinc-finger region with high similarity in the N-terminal transcriptional activation domain. SpYY1 demonstrated identical DNA- and RNA-binding characteristics between Xenopus laevis and S. purpuratus indicating that it maintains similar functional and biochemical properties across widely divergent deuterostome species. SpYY1 binds to the consensus YY1 DNA element, and also to U-rich RNA sequences. Although we detected SpYY1 RNA-binding activity in ova lysates and observed cytoplasmic localization, SpYY1 was not associated with maternal mRNA in ova. SpYY1 expressed in Xenopus oocytes was excluded from the nucleus and associated with maternally expressed cytoplasmic mRNA molecules. These data demonstrate the existence of an YY1 homologue in S. purpuratus with similar structural and biochemical features to those of the well-studied vertebrate YY1; however, the data reveal major differences in the biological role of YY1 in the regulation of maternally expressed mRNA in the two species.

Identification of a novel human estrogen receptor-α splice variant able to enhance malignant biological behaviors of breast cancer cells.

Since the early 1990s, multiple human estrogen receptor-α (hER-α) splice variants have been identified, of which the majority contain ≥1 deleted exon, and some are expressed as proteins with modified functions from the wild-type 66 kDa hER-α (ER-α66). In the present study, a novel hER-α splice variant, ER-α30, was identified and cloned from clinical breast cancer tissue. The ER-α30 sequence lacked a ligand-binding domain and a ligand-dependent transcriptional activation domain but retained the N-terminal transcriptional activation domain, the DNA-binding domain and a partial hinge domain, and possesses a unique 10-amino-acid domain. The expression of ER-α30 was associated with ER-α66-negative and progesterone receptor-negative breast cancer. The 30 kDa protein was expressed in stably transfected MDA-MB-231 cells, and the overexpression of ER-α30 in MDA-MB-231 cells enhanced malignant biological behaviors, including cellular proliferation, migration and invasion in vitro. The results of the present study indicated that ER-α30 might represent a potential biomarker for breast cancer.

Evolution of the POU1F1 transcription factor in mammals: Rapid change of the alternatively-spliced β-domain

The POU1F1 (Pit-1) transcription factor is important in regulating expression of growth hormone, prolactin and TSH β-subunit, and controlling development of the anterior pituitary cells in which these hormones are produced. POU1F1 is a conserved protein comprising three main domains, an N-terminal transcription activation domain (TAD), a POU-specific domain and a C-terminal homeodomain. Within the TAD, a β-domain can be inserted by alternative splicing, giving an extended ‘β-variant’ with altered properties. Here sequence data from over 100 species were used to assess the variability of POU1F1 in mammals. This showed that the POU-specific domain and homeodomain are very strongly conserved, and that the TAD is somewhat less conserved, as are linker and hinge regions between these main domains. On the other hand, the β-domain is very variable, apparently evolving at a rate not significantly different from that expected for unconstrained, neutral evolution. In several species stop and/or frameshift mutations within the β-domain would prevent expression of the β-variant as a functional protein. In most species expression of the β-variant is low (<5% of total POU1F1 expression). The rate of evolution of POU1F1 in mammals shows little variation, though the lineage leading to dog does show an episode of accelerated change. This comparative genomics study suggests that in most mammalian species POU1F1 variants produced by alternative splicing may have little physiological significance.

Comprehensive Identification of Krüppel-Like Factor Family Members Contributing to the Self-Renewal of Mouse Embryonic Stem Cells and Cellular Reprogramming.

Pluripotency is maintained in mouse embryonic stem (ES) cells and is induced from somatic cells by the activation of appropriate transcriptional regulatory networks. Krüppel-like factor gene family members, such as Klf2, Klf4 and Klf5, have important roles in maintaining the undifferentiated state of mouse ES cells as well as in cellular reprogramming, yet it is not known whether other Klf family members exert self-renewal and reprogramming functions when overexpressed. In this study, we examined whether overexpression of any representative Klf family member, such as Klf1–Klf10, would be sufficient for the self-renewal of mouse ES cells. We found that only Klf2, Klf4, and Klf5 produced leukemia inhibitory factor (LIF)-independent self-renewal, although most KLF proteins, if not all, have the ability to occupy the regulatory regions of Nanog, a critical Klf target gene. We also examined whether overexpression of any of Klf1-Klf10 would be sufficient to convert epiblast stem cells into a naïve pluripotent state and found that Klf5 had such reprogramming ability, in addition to Klf2 and Klf4. We also delineated the functional domains of the Klf2 protein for LIF-independent self-renewal and reprogramming. Interestingly, we found that both the N-terminal transcriptional activation and C-terminal zinc finger domains were indispensable for this activity. Taken together, our comprehensive analysis provides new insight into the contribution of Klf family members to mouse ES self-renewal and cellular reprogramming.

Transduction of Recombinant M3-p53-R12 Protein Enhances Human Leukemia Cell Apoptosis.

Tumor suppressor protein p53 plays important roles in initiating cell cycle arrest and promoting tumor cell apoptosis. Previous studies have shown that p53 is either mutated or defective in approximately 50% of human cancers; therefore restoring normal p53 activity in cancer cells might be an effective anticancer therapeutic approach. Herein, we designed a chimeric p53 protein flanked with the MyoD N-terminal transcriptional activation domain (amino acids 1-62, called M3) and a poly-arginine (R12) cell penetrating signal in its N-and C-termini respectively. This chimeric protein, M3-p53-R12, can be expressed in E. coli and purified using immobilized metal ion chromatography followed by serial refolding dialysis. The purified M3-p53-R12 protein retains DNA-binding activity and gains of cell penetrating ability. Using MTT assay, we demonstrated that M3-p53-R12 inhibited the growth of K562, Jurkat as well as HL-60 leukemia cells carrying mutant p53 genes. Results from FACS analysis also demonstrated that transduction of M3-p53-R12 protein induced cell cycle arrest of these leukemia cells. Of special note, M3-p53-R12 has no apoptotic effect on normal mesenchymal stem cells (MSC) and leukocytes, highlighting its differential effects on normal and tumor cells. To sum up, our results reveal that purified recombinant M3-p53-R12 protein has functions of suppressing the leukemia cell lines' proliferation and launching cell apoptosis, suggesting the feasibility of using M3-p53-R12 protein as an anticancer drug. In the future we will test whether this chimeric protein can preferentially trigger the death of malignant cancer cells without affecting normal cells in animals carrying endogenous or xenographic tumors.

MYC interacts with the human STAGA coactivator complex via multivalent contacts with the GCN5 and TRRAP subunits

MYC is an oncogenic DNA-binding transcription activator of many genes and is often upregulated in human cancers. MYC has an N-terminal transcription activation domain (TAD) that is also required for cell transformation. Various MYC TAD-interacting coactivators have been identified, including the transcription/transformation-associated protein (TRRAP), a subunit of different histone acetyltransferase (HAT) complexes such as the human “SPT3–TAF9–GCN5 Acetyltransferase” (STAGA) complex involved in MYC transactivation of the TERT gene. However, it remains unclear whether TRRAP and/or other subunits are directly contacted by MYC within these macromolecular complexes. Here, we characterize the interactions of MYC TAD with the STAGA complex. By protein crosslinking we identify both TRRAP and the GCN5 acetyltransferase as MYC TAD-interacting subunits within native STAGA. We show that purified GCN5 binds to an N-terminal sub-domain of MYC TAD (residues 21–108) and that the interaction of GCN5 and STAGA with this sub-domain is dependent on two related sequence motifs: M2 within the conserved MYC homology box I (MBI), and M3 located between residues 100–106. Interestingly, specific substitutions within the M2/3 motifs that only moderately reduce the intracellular MYC–STAGA interaction and do not influence dimerization of MYC with its DNA-binding partner MAX, strongly inhibit MYC acetylation by GCN5 and reduce MYC binding and transactivation of the GCN5-dependent TERT promoter in vivo. Hence, we propose that MYC associates with STAGA through extended interactions of the TAD with both TRRAP and GCN5 and that the TAD–GCN5 interaction is important for MYC acetylation and MYC binding to certain chromatin loci.

Sequence-specific resonance assignments of human TAF15-RRM and TAF15-RRM-RanBP2

Human TATA binding protein associated factor 2 N (TAF15) is a RNA/DNA binding protein involved in many aspects of RNA and DNA metabolism. TAF15 contains an N-terminal transcriptional activation domain and C-terminal region comprising the RNA recognition motif (RRM) and RanBP2 type zinc finger domains with interspersed RGG motifs. In this study we report the complete backbone and side chain resonance assignments of human TAF15-RRM and backbone assignments of TAF15-RRM-RanBP2.

Structural basis of CBP/p300 recruitment in leukemia induction by E2A-PBX1

AbstractE-proteins are critical transcription factors in B-cell lymphopoiesis. E2A, 1 of 3 E-protein–encoding genes, is implicated in the induction of acute lymphoblastic leukemia through its involvement in the chromosomal translocation 1;19 and consequent expression of the E2A-PBX1 oncoprotein. An interaction involving a region within the N-terminal transcriptional activation domain of E2A-PBX1, termed the PCET motif, which has previously been implicated in E-protein silencing, and the KIX domain of the transcriptional coactivator CBP/p300, critical for leukemogenesis. However, the structural details of this interaction remain unknown. Here we report the structure of a 1:1 complex between PCET motif peptide and the KIX domain. Residues throughout the helical PCET motif that contact the KIX domain are important for both binding KIX and bone marrow immortalization by E2A-PBX1. These results provide molecular insights into E-protein–driven differentiation of B-cells and the mechanism of E-protein silencing, and reveal the PCET/KIX interaction as a therapeutic target for E2A-PBX1–induced leukemia.

E2A proteins enhance the histone acetyltransferase activity of the transcriptional co-activators CBP and p300

The E2A gene encodes the E-protein transcription factors E12 and E47 that play critical roles in B-lymphopoiesis. A somatic chromosomal translocation detectable in 5% of cases of acute lymphoblastic leukemia (ALL) involves E2A and results in expression of the oncogenic transcription factor E2A-PBX1. CREB binding protein (CBP) and its close paralog p300 are transcriptional co-activators with intrinsic histone acetyltransferase (HAT) activity. We and others have shown that direct binding of an N-terminal transcriptional activation domain present in E12/E47 and E2A-PBX1 to the KIX domain of CBP/p300 contributes to E2A protein function. In the current work we show for the first time that the catalytic HAT activity of CBP/p300 is increased in the presence of residues 1–483 of E2A (i.e., the portion present in E2A-PBX1). The addition of purified, recombinant E2A protein to in vitro assays results in a two-fold augmentation of CBP/p300 HAT activity, whereas in vivo assays show a ten-fold augmentation of HAT-dependent transcriptional induction and a five-fold augmentation of acetylation of reporter plasmid-associated histone by CBP in response to co-transfected E2A. Our results indicate that the HAT-enhancing effect is independent of the well-documented E2A–CBP interaction involving the KIX domain and suggest a role for direct, perhaps low affinity binding of E2A to a portion of CBP that includes the HAT domain and flanking elements. Our findings add to a growing body of literature indicating that interactions between CBP/p300 and transcription factors can function in a specific manner to modulate HAT catalytic activity.

Abstract P221: Retinoblastoma Protein--Associated Proteins 48 and 46 Are Novel p300/GATA4-Binding Partners Involved in Hypertrophic Responses in Cardiomyocytes

Background: A zinc finger protein GATA4 is one of hypertrophy-responsive transcription factors, and increases its DNA-binding and transcriptional activities in response to hypertrophic stimuli in cardiomyocytes. Activation of GATA4 during this process is mediated, in part, through acetylation by intrinsic histone acetyltransferases such as a transcriptional coactivator p300. Here, we show that retinoblastoma protein (Rb)-associated protein 48 and 46 (RbAp48, RbAp46), components of NuRD (nucleosome remodeling and deacetylase) complex that has been implicated in chromatin remodeling and transcriptional repression associated with histone deacetylation, are novel components of p300/GATA4 complex. However, the precise functional relationships among p300, GATA4, RbAp48, and RbAp46 remain unknown. Methods and Results: A series of GST pull-down assays revealed that the C-terminal domain of RbAp48/46 bound to the N-terminal transcriptional activation domain of GATA4 and C/H-3 domain of p300, respectively. Immunoprecipitation followed by western blotting demonstrated that RbAp48/46 repressed p300-induced acetylation of GATA4 and histones. While overexpressions of RbAp48/46 inhibited p300/GATA4-induced atrial natriuretic factors (ANF) and endotheline-1 (ET-1) promoter activities, knockdown of neither RbAp48 nor RbAp46 by RNAi enhanced these promoter activities in HEK293 cells. Stimulation of cardiomyocytes with phenylephrine (PE) decreased the binding of GATA4/p300 with RbAp48/46. RbAp48/46 repressed PE-induced hypertrophic responses such as myofibrillar organization, increase in cell size and promoter activation of the ANF and ET-1 in cardiomyocytes. Conclusion: These findings demonstrate that RbAp48 and RbAp46 form a functional protein complex with GATA4/p300 and regulated hypertrophic responses in cardiomyocytes.

Molecular characterization of an EWSR1–POU5F1 fusion associated with a t(6;22) in an undifferentiated soft tissue sarcoma

We report a soft tissue sarcoma from the thigh with morphologic features resembling Ewing sarcoma, clear cell sarcoma, and myoepithelial tumor of soft tissue. In addition, the genetic and immunohistochemical findings do not correspond to any established pattern, so the tumor does not clearly fit into any one classification. The karyotype analysis revealed a rare chromosomal rearrangement, t(6;22)(p22;q12), that previously has been reported in bone and epithelial tumors. Molecular studies confirmed the presence of an EWSR1-POU5F1 fusion creating a chimeric gene with the N-terminal transcriptional activation domain of EWSR1 and the C-terminal POU DNA binding domain of POU5F1. This report is novel in that to our knowledge, it is the first complete molecular characterization of an EWSR1-POU5F1 fusion in a soft tissue sarcoma. Evaluation of existing data on the known EWSR1-POU5F1 tumors suggests that the fusion gene functions in a wide variety of cell types and may modify the differentiation state of cells, resulting in susceptibility to tumorigenesis.

Identification of Ewing’s sarcoma protein as a G‐quadruplex DNA‐ and RNA‐binding protein

The Ewing's sarcoma (EWS) oncogene contains an N-terminal transcription activation domain and a C-terminal RNA-binding domain. Although the EWS activation domain is a potent transactivation domain that is required for the oncogenic activity of several EWS fusion proteins, the normal role of intact EWS is poorly characterized because little is known about its nucleic acid recognition specificity. Here we show that the Arg-Gly-Gly (RGG) domain of the C-terminal in EWS binds to the G-rich single-stranded DNA and RNA fold in the G-quadruplex structure. Furthermore, inhibition of DNA polymerase on a template containing a human telomere sequence in the presence of RGG occurs in an RGG concentration-dependent manner by the formation of a stabilized G-quadruplex DNA-RGG complex. In addition, mutated RGG containing Lys residues replacing Arg residues at specific Arg-Gly-Gly sites and RGG containing Arg methylated by protein arginine N-methyltransferase 3 decrease the binding ability of EWS to G-quadruplex DNA and RNA. These findings suggest that the RGG of EWS binds to G-quadruplex DNA and RNA via the Arg residues in it.

Development of a Mouse Model of DS Megakaryoblastic Leukemia and Identification of the Hsa21 Gene DYRK1A as a Prominent Contributor to DS-AMKL

AbstractAbstract 469Children with Down syndrome (DS) have a 500-fold increased risk of Acute Megakaryoblastic Leukemia (DS-AMKL). In addition to trisomy 21, DS-AMKL blasts harbor somatic mutations in the gene encoding GATA-1, an essential transcriptional regulator of erythroid and megakaryocytic differentiation. In every case, the GATA1 mutations lead to a block in expression of the full-length protein, but allow for the translation of a shortened isoform named GATA-1s, which lacks the N-terminal transcriptional activation domain. Recent studies have shown that mutations in of JAK2, JAK3 and MPL are another feature of AMKL. Whereas the impact on hematopoiesis of individual GATA-1s, JAK2 V617F and MPL W515L mutants is relatively well known, the specific effects of trisomy 21, the initiating event in DS leukemogenesis, remains unclear. Here, we used two different experimental models to identify specific genes on human chromosome 21 (Hsa21) that participate in leukemogenesis: human DS-AMKL cell lines and the Ts1Rhr mouse strain, which is trisomic for only 33 orthologs of Hsa21 genes. Comprehensive analysis of hematopoiesis in progressively more complex mouse models of DS disorders, including Ts1Rhr mice, Ts1Rhr/KI-Gata-1s compound mutant mice, and recipients of Ts1Rhr/KI-Gata-1s hematopoietic progenitors expressing MPL W515L, revealed that the presence of trisomy 21 impacts the phenotype of each genetic background. Ts1Rhr mice develop a progressive myeloproliferative disease with megakaryocytic features while Ts1Rhr/KI-Gata1s double transgenic mice develop thrombocytosis at an earlier age compared to Gata1s mutant or Ts1Rhr mice alone. However neither the Ts1Rhr nor the compound mutant mice develop AMKL. In stark contrast, expression of MPL W515L in Ts1Rhr/KI-Gata1s hematopoietic progenitors led to a rapid lethal megakaryoblastic disorder characterized by profound marrow and splenic fibrosis and the presence of immature megakaryoblasts in lethally irradiated recipients. Of note, transplantation of MPL W515L expressing singly mutant Ts1Rhr or KI-Gata-1s progenitors failed to cause a similar disease. These findings show that three oncogenic events are necessary and sufficient to induce a megakaryoblastic leukemia in recipient mice. In parallel, to identify specific genes on Hsa21 that contribute to AMKL, we performed shRNA screening in DS-AMKL cell lines by knocking down the human orthologs of the 33 genes trisomic in the Ts1Rhr mice. We then evaluated the consequence of knockdown on megakaryocyte proliferation, survival and/or differentiation. This study revealed that altered expression of ERG, DYRK1A, CHAF1B and HLCS affected megakaryocyte growth. Analysis of their expression in human samples suggest that these four genes likely play a role in etiology of DS-AMKL. Moreover expression evaluation of those genes in our in vivo models of 1, 2 or 3 genetic events revealed that ERG, a known megakaryoblastic oncogene, and DYRK1A are the most prominent genes implicated in these megakaryocytic disorders. DYRK1A (dual specificity tyrosine-regulated kinase 1A) has been functionally implicated in many developmental disorders associated with DS and shown to be constitutively phosphorylated/activated in human AMKL cell lines. We found that overexpression of DYRK1A cooperates with Gata1s to lead to a increase in megakaryocyte growth in liquid culture and in colony assays in vitro. In summary, we show that partially trisomic Ts1Rhr mice display progressive defects in the megakaryocytic compartment, that mutagenesis of Gata1 in the Ts1Rhr background exacerbates the Ts1Rhr phenotype, and that addition of an activating mutation of MPL leads to a fulminant megakaryocytic leukemia with myelofibrosis. Moreover, among the 33 genes present in 3 copies in the Ts1Rhr background, we identify DYRK1A as a likely megakaryoblastic oncogene in DS-AMKL. Disclosures:No relevant conflicts of interest to declare.

Inhibitory effect of MIL glycoprotein on expression of pro‐inflammatory mediators in carbon tetrachloride‐induced mice liver damage

The aim of the present study was to evaluate immunomodulatory and hepatoprotective effects of glycoprotein isolated from Morus indica Linne (MIL glycoprotein) on carbon tetrachloride (CCl(4) )-induced liver injury. In the present study, MIL glycoprotein (5 and 10 mg kg(-1) body weight) was administered to ICR mice for 7 days prior to CCl(4) treatment. We evaluated the activities of alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and thiobarbituric acid-reactive substances (TBARS), and expression of inflammation-related mediators including cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-1 beta in CCl(4) -treated mice. Our results revealed that MIL glycoprotein reduced the activities of ALT, LDH and TBARS in serum from CCl(4) -treated mice. We also found that MIL glycoprotein reduced the activity of COX-2 and expression of TNF-α and IL-1 beta in liver from CCl(4) -treated mice. Moreover, administration of MIL glycoprotein suppressed on stress-activated protein kinase/c-jun N-terminal kinase phosphorylation and activator protein-1 transcriptional activation in livers from CCl(4) -treated mice. The results from these experiments indicate that MIL glycoprotein effectively protects against liver injury, mainly through downregulation of oxidative stress and the inflammatory response. In conclusion, we suggest that the MIL glycoprotein might be one component of health supplements for prevention of liver diseases.

Acetylation Directs Survivin Nuclear Localization to Repress STAT3 Oncogenic Activity

The multiple functions of the oncofetal protein survivin are dependent on its selective expression patterns within immunochemically distinct subcellular pools. The mechanism by which survivin localizes to these compartments, however, is only partly understood. Here we show that nuclear accumulation of survivin is promoted by CREB-binding protein (CBP)-dependent acetylation on lysine 129 (129K, Lys-129). We demonstrate a mechanism by which survivin acetylation at this position results in its homodimerization, while deacetylation promotes the formation of survivin monomers that heterodimerize with CRM1 and facilitate its nuclear export. Using proteomic analysis, we identified the oncogenic transcription factor STAT3 as a binding partner of nuclear survivin. We show that acetylated survivin binds to the N-terminal transcriptional activation domain of the STAT3 dimer and represses STAT3 transactivation of target gene promoters. Using multiplex PCR and DNA sequencing, we identified a single-nucleotide polymorphism (A → G) at Lys-129 that exists as a homozygous mutation in a neuroblastoma cell line and corresponds with a defect in survivin nuclear localization. Our results demonstrate that the dynamic equilibrium between survivin acetylation and deacetylation at amino acid 129 determines its interaction with CRM1, its subsequent subcellular localization, and its ability to inhibit STAT3 transactivation, providing a potential route for therapeutic intervention in STAT3-dependent tumors.

Susceptibility of p53 Unstructured N Terminus to 20 S Proteasomal Degradation Programs the Stress Response

The N-terminal transcription activation domain of p53 is intrinsically unstructured. We show in vitro and in vivo that this domain initiates p53 degradation by the 20 S proteasome in a ubiquitin-independent fashion. The decay of metabolically labeled p53 follows biphasic kinetics with an immediate fast phase that is ubiquitin-independent and a second slower phase that is ubiquitin-dependent. The 20 S proteasome executes the first phase by default, whereas the second phase requires the 26 S proteasome. p53 N-terminal binding proteins, such as Hdmx, can selectively block the first phase of degradation. Remarkably, gamma-irradiation inhibits both p53 decay phases, whereas UV selectively negates the second phase, giving rise to discrete levels of p53 accumulation. Our data of a single protein experiencing double mode degradation mechanisms each with unique kinetics provide the mechanistic basis for programmable protein homeostasis (proteostasis).