Search

Ovarian development was traditionally recognized as a "default" sexual outcome and therefore received much less scientific attention than testis development. In turtles with temperature-dependent sex determination (TSD), how the female pathway is initiated to induce ovary development remains unknown. In this study, we have found that phosphorylation of the signal transducer and activator of transcription 3 (pSTAT3) and Foxl2 exhibit temperature-dependent sexually dimorphic patterns and tempo-spatial coexpression in early embryos of the red-eared slider turtle (Trachemys scripta elegans). Inhibition of pSTAT3 at a female-producing temperature of 31 °C induces 64.7% female-to-male sex reversal, whereas activation of pSTAT3 at a male-producing temperature of 26 °C triggers 75.6% male-to-female sex reversal. In addition, pSTAT3 directly binds to the locus of the female sex-determining gene Foxl2 and promotes Foxl2 transcription. Overexpression or knockdown of Foxl2 can rescue the sex reversal induced by inhibition or activation of pSTAT3. This study has established a direct genetic link between warm temperature-induced STAT3 phosphorylation and female pathway initiation in a TSD system, highlighting the critical role of pSTAT3 in the cross talk between female and male pathways. [ABSTRACT FROM AUTHOR]

Lung adenocarcinoma (LUAD) is the leading cause of cancer-related death worldwide, but the underlying molecular mechanisms remain largely unclear. The transcription factor (TF) specificity protein 1 (SP1) plays a crucial role in the development of various cancers, including LUAD. Recent studies have indicated that master TFs may form phase-separated macromolecular condensates to promote super-enhancer (SE) assembly and oncogene expression. In this study, we demonstrated that SP1 undergoes phase separation and that its zinc finger 3 in the DNA-binding domain is essential for this process. Through Cleavage Under Targets & Release Using Nuclease (CUT&RUN) using antibodies against SP1 and H3K27ac, we found a significant correlation between SP1 enrichment and SE elements, identified the regulator of the G protein signaling 20 (RGS20) gene as the most likely target regulated by SP1 through SE mechanisms, and verified this finding using different approaches. The oncogenic activity of SP1 relies on its phase separation ability and RGS20 gene activation, which can be abolished by glycogen synthase kinase J4 (GSK-J4), a demethylase inhibitor. Together, our findings provide evidence that SP1 regulates its target oncogene expression through phase separation and SE mechanisms, thereby promoting LUAD cell progression. This study also revealed an innovative target for LUAD therapies through intervening in SP1-mediated SE formation. [ABSTRACT FROM AUTHOR]

Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many disorders including cardiac arrhythmias. Prior work revealed association of mRNAs encoding cardiac NaV1.5 (SCN5A) and hERG1 (KCNH2), but the functional significance of this association was not established. Here, we provide a more comprehensive picture of KCNH2, SCN5A, CACNA1C, and KCNQ1 transcripts collectively copurifying with nascent hERG1, NaV1.5, CaV1.2, or KCNQ1 channel proteins. Single-molecule fluorescence in situ hybridization (smFISH) combined with immunofluorescence reveals that the channel proteins are synthesized predominantly as heterotypic pairs from discrete molecules of mRNA, not as larger cotranslational complexes. Puromycin disrupted colocalization of mRNA with its encoded protein, as expected, but remarkably also pairwise mRNA association, suggesting that transcript association relies on intact translational machinery or the presence of the nascent protein. Targeted depletion of KCHN2 by specific shRNA resulted in concomitant reduction of all associated mRNAs, with a corresponding reduction in the encoded channel currents. This co-knockdown effect, originally described for KCNH2 and SCN5A, thus appears to be a general phenomenon among transcripts encoding functionally related proteins. In multielectrode array recordings, proarrhythmic behavior arose when IKr was reduced by the selective blocker dofetilide at IC50 concentrations, but not when equivalent reductions were mediated by shRNA, suggesting that co-knockdown mitigates proarrhythmic behavior expected from the selective reduction of a single channel species. We propose that coordinated, cotranslational association of functionally related ion channel mRNAs confers electrical stability by co-regulating complementary ion channels in macromolecular complexes. [ABSTRACT FROM AUTHOR]

Endothelial cell (EC) sensing of wall fluid shear stress (FSS) from blood flow governs vessel remodeling to maintain FSS at a specific magnitude or set point in healthy vessels. Low FSS triggers inward remodeling to restore normal FSS but the regulatory mechanisms are unknown. In this paper, we describe the signaling network that governs inward artery remodeling. FSS induces Smad2/3 phosphorylation through the type I transforming growth factor (TGF)-β family receptor Alk5 and the transmembrane protein Neuropilin-1, which together increase sensitivity to circulating bone morphogenetic protein (BMP)-9. Smad2/3 nuclear translocation and target gene expression but not phosphorylation are maximal at low FSS and suppressed at physiological high shear. Reducing flow by carotid ligation in rodents increases Smad2/3 nuclear localization, while the resultant inward remodeling is blocked by the EC-specific deletion of Alk5. The flow-activated MEKK3/Klf2 pathway mediates the suppression of Smad2/3 nuclear translocation at high FSS, mainly through the cyclin-dependent kinase (CDK)-2-dependent phosphosphorylation of the Smad linker region. Thus, low FSS activates Smad2/3, while higher FSS blocks nuclear translocation to induce inward artery remodeling, specifically at low FSS. These results are likely relevant to inward remodeling in atherosclerotic vessels, in which Smad2/3 is activated through TGF-ß signaling. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies [A. M. Valdes, T. D. Spector, Nat. Rev. Rheumatol. 7, 23-32 (2011)]. We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury [W. Lee et al., Proc. Natl. Acad. Sci. U.S.A. 111, E5114-E5122 (2014); W. Lee, F. Guilak, W. Liedtke, Curr. Top. Membr. 79, 263-273 (2017)]. We therefore asked whether interleukin-1-mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1a (IL-1a) up-regulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca2+ at baseline and in response to mechanical deformation. Elevated resting state Ca2+ in turn rarefied the F-actin cytoskeleton and amplified mechanically induced deformation microtrauma. As intracellular substrates of this OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1a, we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. Taken together, these signaling and genetic reprogramming events represent a detrimental Ca2+-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA. [ABSTRACT FROM AUTHOR]

Rechargeability and operational safety of commercial lithium (Li)- ion batteries demand further improvement. Plating of metallic Li on graphite anodes is a critical reason for Li-ion battery capacity decay and short circuit. It is generally believed that Li plating is caused by the slow kinetics of graphite intercalation, but in this paper, we demonstrate that thermodynamics also serves a crucial role. We show that a nonuniform temperature distribution within the battery can make local plating of Li above 0 V vs. Li0/Li+ (room temperature) thermodynamically favorable. This phenomenon is caused by temperature-dependent shifts of the equilibrium potential of Li0/Li+. Supported by simulation results, we confirm the likelihood of this failure mechanism during commercial Li-ion battery operation, including both slow and fast charging conditions. This work furthers the understanding of nonuniform Li plating and will inspire future studies to prolong the cycling lifetime of Li-ion batteries. [ABSTRACT FROM AUTHOR]

The aberrant hedgehog (Hh) pathway plays important roles in multiple cancer types, therefore serving as a promising drug target. Current clinically available hedgehog-targeted drugs act mostly by antagonizing the upstream component smoothened; however, both primary and acquired resistance to FDA-approved smoothened inhibitor (SMOi) drugs have been described. We have recently demonstrated that the BET inhibitor effectively suppresses SMOiresistant Hh-driven cancers through antagonizing transcription of GLI1 and GLI2, the core transcriptional factors of Hh pathway, suggesting epigenetic or transcriptional targeted therapy represents an anti-Hh therapeutic strategy that can overcome SMOi resistance. Here we performed an unbiased screening of epigenetic or transcriptional targeted small molecules to test their inhibitory effects on GLI1 and GLI2 transcription or cell viability of Hh-driven tumor lines. THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7), is identified as the top hit in our screening. We then confirmed that antagonizing CDK7 by either small-molecule inhibitors or the CRISPR-Cas9 approach causes substantial suppression of GLI1 and GLI2 transcription, resulting in effective inhibition of Hh-driven cancers in vitro and in vivo. More importantly, antagonizing CDK7 retains inhibitory activity against Hh-driven cancers with almost all sofar described primary or acquired SMOi resistance. Furthermore, we reveal a synergy between CDK7 inhibition and BET inhibition on antagonizing aberrant Hh pathway and Hh-driven cancers that are either responsive or resistant to SMOi. Our results illustrate transcriptional inhibition through targeting CDK7 as a promising therapeutic strategy for treating Hh-driven cancers, especially those with primary or acquired resistance to SMOi drugs. [ABSTRACT FROM AUTHOR]

Relapsed and refractory (R/R) multiple myeloma (MM) patients have very poor prognosis. Chimeric antigen receptor modified T (CAR T) cells is an emerging approach in treating hematopoietic malignancies. Here we conducted the clinical trial of a biepitope-targeting CAR T against B cell maturation antigen (BCMA) (LCAR-B38M) in 17 R/R MM cases. CAR T cells were i.v. infused after lymphodepleting chemotherapy. Two delivery methods, three infusions versus one infusion of the total CAR T dose, were tested in, respectively, 8 and 9 cases. No response differences were noted among the two delivery subgroups. Together, after CART cell infusion, 10 cases experienced a mild cytokine release syndrome (CRS), 6 had severe but manageable CRS, and 1 died of a very severe toxic reaction. The abundance of BCMA and cytogenetic marker del(17p) and the elevation of IL-6 were the key indicators for severe CRS. Among 17 cases, the overall response rate was 88.2%, with 13 achieving stringent complete response (sCR) and 2 reaching very good partial response (VGPR), while 1 was a nonresponder. With a median follow-up of 417 days, 8 patients remained in sCR or VGPR, whereas 6 relapsed after sCR and 1 had progressive disease (PD) after VGPR. CAR T cells were high in most cases with stable response but low in 6 out of 7 relapse/PD cases. Notably, positive anti-CAR antibody constituted a high-risk factor for relapse/PD, and patients who received prior autologous hematopoietic stem cell transplantation had more durable response. Thus, biepitopic CAR T against BCMA represents a promising therapy for R/R MM, while most adverse effects are clinically manageable. [ABSTRACT FROM AUTHOR]

Neurotrophic factor NRG1 and its receptor ErbB4 play a role in GABAergic circuit assembly during development. ErbB4 null mice possess fewer interneurons, have decreased GABA release, and show impaired behavior in various paradigms. In addition, NRG1 and ErbB4 have also been implicated in regulating GABAergic transmission and plasticity in matured brains. However, current ErbB4 mutant strains are unable to determine whether phenotypes in adult mutant mice result from abnormal neural development. This important question, a glaring gap in understanding NRG1-ErbB4 function, was addressed by using two strains of mice with temporal control of ErbB4 deletion and expression, respectively. We found that ErbB4 deletion in adult mice impaired behavior and GABA release but had no effect on neuron numbers and morphology. On the other hand, some deficits due to the ErbB4 null mutation during development were alleviated by restoring ErbB4 expression at the adult stage. Together, our results indicate a critical role of NRG1-ErbB4 signaling in GABAergic transmission and behavior in adulthood and suggest that restoring NRG1- ErbB4 signaling at the postdevelopmental stage might benefit relevant brain disorders. [ABSTRACT FROM AUTHOR]

Most B cell precursor acute lymphoblastic leukemia (BCP ALL) can be classified into known major genetic subtypes, while a substantial proportion of BCP ALL remains poorly characterized in relation to its underlying genomic abnormalities. We therefore initiated a large-scale international study to reanalyze and delineate the transcriptome landscape of 1,223 BCP ALL cases using RNA sequencing. Fourteen BCP ALL gene expression subgroups (G1 to G14) were identified. Apart from extending eight previously described subgroups (G1 to G8 associated with MEF2D fusions, TCF3-PBX1 fusions, ETV6-RUNX1-positive/ETV6-RUNX1-like, DUX4 fusions, ZNF384 fusions, BCR-ABL1/Ph-like, high hyperdiploidy, and KMT2A fusions), we defined six additional gene expression subgroups: G9 was associated with both PAX5 and CRLF2 fusions; G10 and G11 with mutations in PAX5 (p.P80R) and IKZF1 (p.N159Y), respectively; G12 with IGH-CEBPE fusion and mutations in ZEB2 (p.H1038R); and G13 and G14 with TCF3/4-HLF and NUTM1 fusions, respectively. In pediatric BCP ALL, subgroups G2 to G5 and G7 (51 to 65/67 chromosomes) were associated with low-risk, G7 (with ≤50 chromosomes) and G9 were intermediate-risk, whereas G1, G6, and G8 were defined as high-risk subgroups. In adult BCP ALL, G1, G2, G6, and G8 were associated with high risk, while G4, G5, and G7 had relatively favorable outcomes. This large-scale transcriptome sequence analysis of BCP ALL revealed distinct molecular subgroups that reflect discrete pathways of BCP ALL, informing disease classification and prognostic stratification. The combined results strongly advocate that RNA sequencing be introduced into the clinical diagnostic workup of BCP ALL. [ABSTRACT FROM AUTHOR]

Neuregulin3 (NRG3) is a growth factor of the neuregulin (NRG) family and a risk gene of various severe mental illnesses including schizophrenia, bipolar disorders, and major depression. However, the physiological function of NRG3 remains poorly understood. Here we show that loss of Nrg3 in GFAP-Nrg3f/f mice increased glutamatergic transmission, but had no effect on GABAergic transmission. These phenotypes were observed in Nex-Nrg3f/f mice, where Nrg3 was specifically knocked out in pyramidal neurons, indicating that Nrg3 regulates glutamatergic transmission by a cell-autonomous mechanism. Consequently, in the absence of Nrg3 in pyramidal neurons, mutant mice displayed various behavioral deficits related to mental illnesses. We show that the Nrg3 mutation decreased paired-pulse facilitation, increased decay of NMDAR currents when treated with MK801, and increased minimal stimulation-elicited response, providing evidence that the Nrg3 mutation increases glutamate release probability. Notably, Nrg3 is a presynaptic protein that regulates the SNARE-complex assembly. Finally, increased Nrg3 levels, as observed in patients with severe mental illnesses, suppressed glutamatergic transmission. Together, these observations indicate that, unlike the prototype Nrg1, the effect of which is mediated by activating ErbB4 in interneurons, Nrg3 is critical in controlling glutamatergic transmission by regulating the SNARE complex at the presynaptic terminals, identifying a function of Nrg3 and revealing a pathophysiological mechanism for hypofunction of the glutamatergic pathway in Nrg3-related severe mental illnesses. [ABSTRACT FROM AUTHOR]

Oligomers of homomeric voltage-gated potassium channels associate early in biogenesis as the nascent proteins emerge from the polysome. Less is known about howproteins emerging fromdifferent polysomes associate to form hetero-oligomeric channels. Here, we report that alternate mRNA transcripts encoding human ether-à-go-go-related gene (hERG) 1a and 1b subunits, which assemble to produce ion channels mediating cardiac repolarization, are physically associated during translation. We show that shRNA specifically targeting either hERG 1a or 1b transcripts reduced levels of both transcripts, but only when they were coexpressed heterologously. Both transcripts could be copurified with an Ab against the nascent hERG 1a N terminus. This interaction occurred even when translation of 1b was prevented, indicating the transcripts associate independent of their encoded proteins. The association was also demonstrated in cardiomyocytes, where levels of both hERG transcripts were reduced by either 1a or 1b shRNA, but native KCNE1 and ryanodine receptor 2 (RYR2) transcripts were unaffected. Changes in protein levels and membrane currents mirrored changes in transcript levels, indicating the targeted transcripts were undergoing translation. The physical association of transcripts encoding different subunits provides the spatial proximity required for nascent proteins to interact during biogenesis, and may represent a general mechanism facilitating assembly of heteromeric protein complexes involved in a range of biological processes. [ABSTRACT FROM AUTHOR]

Intensive rice breeding over the past 50 y has dramatically increased productivity especially in the indica subspecies, but our knowledge of the genomic changes associated with such improvement has been limited. In this study, we analyzed low-coverage sequencing data of 1,479 rice accessions from 73 countries, including landraces and modern cultivars. We identified two major subpopulations, indica I (IndI) and indica II (IndII), in the indica subspecies, which corresponded to the two putative heterotic groups resulting from independent breeding efforts. We detected 200 regions spanning 7.8% of the rice genome that had been differentially selected between IndI and IndII, and thus referred to as breeding signatures. These regions included large numbers of known functional genes and loci associated with important agronomic traits revealed by genome-wide association studies. Grain yield was positively correlated with the number of breeding signatures in a variety, suggesting that the number of breeding signatures in a line may be useful for predicting agronomic potential and the selected loci may provide targets for rice improvement. [ABSTRACT FROM AUTHOR]

The human ether-à-go-go-related gene (hERG; or KCNH2) encodes the voltage-gated potassium channel underlying IKr, a repolarizing current in the heart. Mutations in KCNH2 or pharmacological agents that reduce IKr slow action potential (AP) repolarization and can trigger cardiac arrhythmias associated with long QT syndrome. Two channel-forming subunits encoded by KCNH2 (hERG 1a and 1b) are expressed in cardiac tissue. In heterologous expression systems, these subunits avidly coassemble and exhibit biophysical and pharmacological properties distinct from those of homomeric hERG 1a channels. Despite these findings, adoption of hERG 1a/1b hetero-meric channels as a model for cardiac IKr has been hampered by the lack of evidence for a direct functional role for the 1b subunit in native tissue. In this study, we measured IKr and APs at physiological temperature in cardiomyocytes derived from human induced plurip-otent stem cells (iPSC-CMs). We found that specific knockdown of the 1b subunit using shRNA caused reductions in 1b mRNA, 1b protein levels, and IKr magnitude by roughly one-half. AP duration was increased and AP variability was enhanced relative to controls. Early afterdepolarizations, considered cellular substrates for arrhythmia, were also observed in cells with reduced 1b expression. Similar behavior was elicited when channels were effectively converted from heteromers to 1a homomers by expressing a fragment corresponding to the 1a-specific N-terminal Per-Arnt-Sim domain, which is omitted from hERG 1b by alternate transcription. These findings establish that hERG 1b is critical for normal repolarization and that loss of 1b is proarrhythmic in human cardiac cells. [ABSTRACT FROM AUTHOR]

Endothelial cells (ECs) express fibroblast growth factor receptors (FGFRs) and are exquisitely sensitive to FGF signals. However, whether the EC or another vascular cell type requires FGF signaling during development, homeostasis, and response to injury is not known. Here, we show that Flk1-Cre or Tie2-Cre mediated deletion of FGFR1 and FGFR2 {Fgfr½Flk1-Cre or FgfrV 2tie2-cre mice), which results in deletion in endothelial and hematopoietic cells, is compatible with normal embryonic development. As adults, Fgfr½Flk-1-Cre mice maintain normal blood pressure and vascular reactivity and integrity under homeostatic conditions. However, neovascularization after skin or eye injury was significantly impaired in both Fgfr½Flk-1-Cre and Fgfr½½Flk-1-CreTie2-Cre½Flk-1-Cre mice, independent of either hematopoietic cell loss of FGFR½ or vascular endothelial growth factor receptor 2 (Vegfr2) haploinsuf-ficiency. Also, impaired neovascularization was associated with delayed cutaneous wound healing. These findings reveal a key requirement for cell-autonomous EC FGFR signaling in injury-induced angiogenesis, but not for vascular homeostasis, identifying the EC FGFR signaling pathway as a target for diseases associated with aberrant vascular proliferation, such as age-related macular degeneration, and for modulating wound healing without the potential toxicity associated with direct manipulation of systemic FGF or VEGF activity. [ABSTRACT FROM AUTHOR]

A posttranslational protein O-mannosylation process resembling that found in fungi and animals has been reported in the major human pathogen Mycobacterium tuberculosis (Mtb) and related actinobacterja. However, the role and incidence of this process, which is essential in eukaryotes, have never been explored in Mtb. We thus analyzed the impact of interrupting O-mannosylation in the nonpathogenic saprophyte Mycobacterium smegmatis and in the human pathogen Mtb by inactivating the respective putative protein mannosyl transferase genes Msmeg_5447 and RvlOO2c. Loss of protein O-mannosylation in both mutant strains was unambiguously demonstrated by efficient mass spectrometry-based glycoproteomics analysis. Unexpectedly, although the M. smegmatis phenotype was unaffected by the lack of man no-proteins, the Mtb mutant had severely impacted growth in vitro and in cellulo associated with a strong attenuation of its pathogenicity in immunocompromised mice. These data are unique in providing evidence of the biological significance of protein O-mannosylation in mycobacteria and demonstrate the crucial contribution of this protein posttranslational modification to Mtb virulence in the host. [ABSTRACT FROM AUTHOR]

MicroRNA 21 (miR-21) is overexpressed in virtually all types of carcinomas and various types of hematological malignancies. To determine whether miR-21 promotes tumor development in vivo, we knocked out the miR-21 allele in mice. In response to the 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate mouse skin carcinogenesis protocol, miR-21-null mice showed a significant reduction in papilloma formation compared with wild-type mice. We revealed that cellular apoptosis was elevated and cell proliferation was decreased in mice deficient of miR-21 compared to wild-type animals. In addition, we found that a large number of validated or predicted miR-21 target genes were up-regulated in miR-21-null keratinocytes, which are precursor cells to skin papillomas. Specifically, up-regulation of Spry1, Pten, and Pdcd4 when miR-21 was ablated coincided with reduced phosphorylation of ERK, AKT, and JNK, three major downstream effectors of Ras activation that plays a predominant role in DMBA-initiated skin carcinogenesis. These results provide in vivo evidence that miR-21 exerts its oncogenic function through negatively regulating its target genes. [ABSTRACT FROM AUTHOR]

All-trans retinoic acid (ATRA)/arsenic trioxide (ATO) combination-based therapy has benefitted newly diagnosed acute promyelocytic leukemia (APL) in short-term studies, but the long-term efficacy and safety remained unclear. From April 2001, we have followed 85 patients administrated ATRA/ATO with a median follow-up of 70 months. Eighty patients (94.1%) entered complete remission (CR). Kaplan-Meier estimates of the 5-year event-free survival (EFS) and overall survival (OS) for all patients were 89.2% ± 3.4% and 91.7% ± 3.0%, respectively, and the 5-year relapse-free survival (RFS) and OS for patients who achieved CR (n = 80) were 94.8% ± 2.5% and 97.4% ± 1.8%, respectively. Upon ATRA/ATO, prognosis was not influenced by initial white blood cell count, distinct PML-RARα types, or FLT3 mutations. The toxicity profile was mild and revers- ible. No secondary carcinoma was observed, and 24 months after the last dose of ATRA/ATO, patients had urine arsenic concentrations well below the safety limit. These results demonstrate the high efficacy and minimal toxicity of ATRA/ATO treatment for newly diagnosed APL in long-term follow-up, suggesting a potential frontline therapy for de novo APL. [ABSTRACT FROM AUTHOR]

Bmi-1 and SALL4 are putative oncogenes that modulate stem cell pluripotency and play a role in leukemogenesis. Murine Sail4 also has been shown to play an essential role in maintaining the properties of ES cells and governing the fate of the primitive inner cell mass. Here, we demonstrate that transcription from the Bmi-1 promoter is strikingly activated by SALL4 in a close-dependent manner by using a luciferase reporter gene assay. Both promoter deletion construct studies and ChIP from a myeloid stem cell line, 32D, demonstrate that SALL4 binds to a specific region of the Bmi-1 promoter. Deletion of one copy of Sall4 by gene targeting in mouse bone marrow significantly reduced Bmi-1 expression. Reducing SALL4 expression by siRNA in the HL-60 leukemia cell line also results in significant down-regulation of Bmi-1. Furthermore, Bmi-1 expression is up-regulated in transgenic mice that constitutively overexpress human SALL4, and the levels of Bmi-1 in these mice increase as they progress from normal to preleukemic (myelodysplastic syndrome) and leukemic (acute myeloid leukemia) stages. High levels of H3-K4 trimethylation and H3-K79 dimethylation were observed in the SALL4 binding region of the Bmi-1 promoter. These findings suggest a novel link between SALL4 and Bmi-1 in regulating self-renewal of normal and leukemic stem cells. An increase in histone H3-K4 and H3-K79 methylation within the Bmi-1 promoter provides an epigenetic mechanism for histone modifications in SALL4-mediated Bmi-1 gene deregulation. [ABSTRACT FROM AUTHOR]

Smad proteins mediate transforming growth factor β (TGF-β)inducible transcriptional responses. Protein inhibitor of activated signal transducer and activator of transcription (PIAS) represents a family of proteins that inhibits signal transducer and activator of transcription and also regulates other transcriptional responses. In an effort to identify Smad-interacting proteins by a yeast threehybrid screen with Smad3 and Smad4 as baits, we identified PIASy, a member of the PIAS family. In yeast, PIASy interacts strongly with Smad4 and also with receptor-regulated Smads. In mammalian cells, PIASy binds most strongly with Smad3 and also associates with other receptor-regulated Smads and Smad4. The interaction between Smad3 and PIASy is increased in the presence of TGF-β and occurs through the C-terminal domain of Smad3. Moreover, Smad3, Smad4, and PIASy can form a ternary complex. PIASy does not inhibit Smad complex binding to DNA, but it represses Smad transcriptional activity. Interestingly, conditional overexpression of PIASy selectively inhibits a subset of endogenous TGF-β-responsive genes, which includes the cyclin-dependent kinase inhibitor p15, and the plasminogen activator inhibitor 1. We further show that PIASy can interact constitutively with histone deacetylase 1 (HDAC1) and that addition of HDAC inhibitor trichostatin A (TSA) can prevent the inhibitory function of PIASy. Taken together, our studies indicate that PIASy can inhibit TGF-β/Smad transcriptional responses through interactions with Smad proteins and HDAC. [ABSTRACT FROM AUTHOR]