Project description:Mutations that affect myelodysplasia/myeloid leukemia factor (MLF) proteins are associated with leukemia and several other cancers. However, with no strong homology to other proteins of known function, the role of MLF proteins in the cell has remained elusive. Here, we describe a proteomics approach that identifies MLF as a member of a nuclear chaperone complex containing a DnaJ protein, BCL2-associated anthanogene 2, and Hsc70. This complex associates with chromatin and regulates the expression of target genes. The MLF complex is bound to sites of nucleosome depletion and sites containing active chromatin marks (e.g., H3K4me3 and H3K4me1). Hence, MLF binding is enriched at promoters and enhancers. Additionally, the MLF-chaperone complex functions to regulate transcription factor stability, including the RUNX transcription factor involved in hematopoiesis. Although Hsc70 and other co-chaperones have been shown to play a role in nuclear translocation of a variety of proteins including transcription factors, our findings suggest that MLF and the associated co-chaperones play a direct role in modulating gene transcription.
Project description:An exciting recent approach to targeting transcription factors in cancer is to block formation of oncogenic complexes. We investigated whether interfering with the interaction of the transcription factor SALL4, which is critical for leukemic cell survival, and its epigenetic partner complex represents a novel therapeutic approach. The mechanism of SALL4 in promoting leukemogenesis is at least in part mediated by its repression of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) through its interaction with a histone deacetylase (HDAC) complex. In this study, we demonstrate that a peptide can compete with SALL4 in interacting with the HDAC complex and reverse its effect on PTEN repression. Treating SALL4-expressing malignant cells with this peptide leads to cell death that can be rescued by a PTEN inhibitor. The antileukemic effect of this peptide can be confirmed on primary human leukemia cells in culture and in vivo, and is identical to that of down-regulation of SALL4 in these cells using an RNAi approach. In summary, our results demonstrate a novel peptide that can block the specific interaction between SALL4 and its epigenetic HDAC complex in regulating its target gene, PTEN. Furthermore, targeting SALL4 with this approach could be an innovative approach in treating leukemia.
Project description:Drosophila myelodysplasia/myeloid leukemia factor (dMLF), a homolog of human MLF1, oncogene was first identified by yeast two-hybrid screen using the DNA replication-related element-binding factor (DREF) as bait. DREF is a transcription factor that regulates proliferation-related genes in Drosophila. It is known that overexpression of dMLF in the wing imaginal discs through the engrailed-GAL4 driver causes an atrophied wing phenotype associated with the induction of apoptosis. However, the precise mechanisms involved have yet to be clarified. Here, we found the atrophied phenotype to be suppressed by loss-of-function mutation of Drosophila Jun N-terminal kinase (JNK), basket (bsk). Overexpression of dMLF induced ectopic JNK activation in the wing disc monitored with the puckered-lacZ reporter line, resulting in induction of apoptosis. The DREF-binding consensus DRE sequence could be shown to exist in the bsk promoter. Chromatin immunoprecipitation assays in S2 cells with anti-dMLF IgG and quantitative real-time PCR revealed that dMLF binds specifically to the bsk promoter region containing the DRE sequence. Furthermore, using a transient luciferase expression assay, we provide evidence that knockdown of dMLF reduced bsk gene promoter activity in S2 cells. Finally, we show that dMLF interacts with DREF in vivo. Altogether, these data indicate that dMLF acts with DREF to stimulate the bsk promoter and consequently activates the JNK pathway to promote apoptosis.
Project description:Acute myeloid leukemia (AML) is a highly heterogeneous cancer associated with different patterns of gene expression determined by the nature of their DNA mutations. These mutations mostly act to deregulate gene expression by various mechanisms at the level of the nucleus. By performing genome-wide epigenetic profiling of cis-regulatory elements, we found that AML encompasses different mutation-specific subclasses associated with the rewiring of the gene regulatory networks that drive differentiation into different directions away from normal myeloid development. By integrating epigenetic profiles with gene expression and chromatin conformation data, we defined pathways within gene regulation networks that were differentially rewired within each mutation-specific subclass of AML. This analysis revealed 2 major classes of AML: one class defined by mutations in signaling molecules that activate AP-1 via the mitogen-activated protein (MAP) kinase pathway and a second class defined by mutations within genes encoding transcription factors such as RUNX1/CBFβ and C/EBPα. By identifying specific DNA motifs protected from DNase I digestion at cis-regulatory elements, we were able to infer candidate transcription factors bound to these motifs. These integrated analyses allowed the identification of AML subtype-specific core regulatory networks that are required for AML development and maintenance, which could now be targeted in personalized therapies.
Project description:RUNX1 is generally considered a tumor suppressor in myeloid neoplasms. Inactivating RUNX1 mutations have frequently been found in patients with myelodysplastic syndrome (MDS) and cytogenetically normal acute myeloid leukemia (AML). However, no somatic RUNX1 alteration was found in AMLs with leukemogenic fusion proteins, such as core-binding factor (CBF) leukemia and MLL fusion leukemia, raising the possibility that RUNX1 could actually promote the growth of these leukemia cells. Using normal human cord blood cells and those expressing leukemogenic fusion proteins, we discovered a dual role of RUNX1 in myeloid leukemogenesis. RUNX1 overexpression inhibited the growth of normal cord blood cells by inducing myeloid differentiation, whereas a certain level of RUNX1 activity was required for the growth of AML1-ETO and MLL-AF9 cells. Using a mouse genetic model, we also showed that the combined loss of Runx1/Cbfb inhibited leukemia development induced by MLL-AF9. RUNX2 could compensate for the loss of RUNX1. The survival effect of RUNX1 was mediated by BCL2 in MLL fusion leukemia. Our study unveiled an unexpected prosurvival role for RUNX1 in myeloid leukemogenesis. Inhibiting RUNX1 activity rather than enhancing it could be a promising therapeutic strategy for AMLs with leukemogenic fusion proteins.
Project description:Acute myeloid leukemia (AML) is an aggressive hematological malignancy with high relapse/refractory rate. Genetic and epigenetic abnormalities are driving factors for leukemogenesis. RUNX1 and RUNX2 from the Runt-related transcription factor (RUNX) family played important roles in AML pathogenesis. However, the relationship between RUNX3 and AML remains unclear. Here, we found that RUNX3 was a super-enhancer-associated gene and highly expressed in AML cells. The Cancer Genome Atlas (TCGA) database showed high expression of RUNX3 correlated with poor prognosis of AML patients. We observed that Runx3 knockdown significantly inhibited leukemia progression by inducing DNA damage to enhance apoptosis in murine AML cells. By chromatin immunoprecipitation sequencing (ChIP-seq) analysis, we discovered that RUNX3 in AML cells mainly bound more genes involved in DNA-damage repair and antiapoptosis pathways compared to that in normal bone marrow cells. Runx3 knockdown obviously inhibited the expression of these genes in AML cells. Overall, we identified RUNX3 as an oncogene overexpressed in AML cells, and Runx3 knockdown suppressed AML progression by inducing DNA damage and apoptosis.
Project description:The heat shock response, resulting in the production of heat shock proteins or molecular chaperones, is triggered by elevated temperature and a variety of other stressors. Its master regulator is heat shock transcription factor 1 (HSF1). Heat shock factors generally exist in multiple isoforms. The two known isoforms of HSF1 differ in the inclusion (HSF1α) or exclusion (HSF1β) of exon 11. Although there are some data concerning the differential expression patterns and transcriptional activities of HSF2 isoforms during development, little is known about the distinct properties of the HSF1 isoforms. Here we present evidence for two novel HSF1 isoforms termed HSF1γα and HSF1γβ, and we show that the HSF1 isoform ratio differentially regulates heat shock protein gene transcription. Hsf1γ isoforms are expressed in various mouse tissues and are translated into protein. Furthermore, after heat shock, HSF1γ isoforms are exported from the nucleus more rapidly or degraded more quickly than HSF1α or HSF1β. We also show that each individual HSF1 isoform is sufficient to induce the heat shock response and that expression of combinations of HSF1 isoforms, in particular HSF1α and HSF1β, results in a synergistic enhancement of the transcriptional response. In addition, HSF1γ isoforms potentially suppress the synergistic effect of HSF1α and HSF1β co-expression. Collectively, our observations suggest that the expression of HSF1 isoforms in a specific ratio provides an additional layer in the regulation of heat shock protein gene transcription.
Project description:Superoxide dismutase 1 (Sod1) has been known for nearly half a century for catalysis of superoxide to hydrogen peroxide. Here we report a new Sod1 function in oxidative signalling: in response to elevated endogenous and exogenous reactive oxygen species (ROS), Sod1 rapidly relocates into the nucleus, which is important for maintaining genomic stability. Interestingly, H2O2 is sufficient to promote Sod1 nuclear localization, indicating that it is responding to general ROS rather than Sod1 substrate superoxide. ROS signalling is mediated by Mec1/ATM and its effector Dun1/Cds1 kinase, through Dun1 interaction with Sod1 and regulation of Sod1 by phosphorylation at S60, 99. In the nucleus, Sod1 binds to promoters and regulates the expression of oxidative resistance and repair genes. Altogether, our study unravels an unorthodox function of Sod1 as a transcription factor and elucidates the regulatory mechanism for its localization.
Project description:Proteins fold under mechanical forces in a number of biological processes, ranging from muscle contraction to co-translational folding. As force hinders the folding transition, chaperones must play a role in this scenario, although their influence on protein folding under force has not been directly monitored yet. Here, we introduce single-molecule magnetic tweezers to study the folding dynamics of protein L in presence of the prototypical molecular chaperone trigger factor over the range of physiological forces (4-10?pN). Our results show that trigger factor increases prominently the probability of folding against force and accelerates the refolding kinetics. Moreover, we find that trigger factor catalyzes the folding reaction in a force-dependent manner; as the force increases, higher concentrations of trigger factor are needed to rescue folding. We propose that chaperones such as trigger factor can work as foldases under force, a mechanism which could be of relevance for several physiological processes.Proteins fold under mechanical force during co-translational folding at the ribosome. Here, the authors use single molecule magnetic tweezers to study the influence of chaperones on protein folding and show that the ribosomal chaperone trigger factor acts as a mechanical foldase by promoting protein folding under force.
Project description:OBJECTIVES:Control of cell proliferation is critical for accurate cell differentiation and tissue formation, during development and regeneration. Here, we have analysed the role of microphthalmia-associated transcription factor MITF and its direct target, T-box factor TBX2, in regulating proliferation of mammalian neural crest-derived melanocytes. MATERIALS AND METHODS:Immunohistochemistry was used to examine spatial and temporal expression of TBX2 in melanocytes in vivo. RNAi and cell proliferation analysis were used to investigate functional roles of TBX2. Furthermore, quantitative RT-PCR, western blot analysis and flow cytometry were used to further scrutinize molecular mechanisms underlying TBX2-dependent cell proliferation. RESULTS:TBX2 was found to be co-expressed with MITF in melanocytes of mouse hair follicles. Specific Tbx2 knockdown in primary neural crest cells led to inhibition MITF-positive melanoblast proliferation. Tbx2 knockdown in melan-a cells led to reduction in Cyclin D1 expression and G1-phase cell cycle arrest. TBX2 directly activated Ccnd1 transcription by binding to a specific sequence in the Ccnd1 promoter, and the defect in cell proliferation could be rescued partially by overexpression of Cyclin D1 in Tbx2 knockdown melanocytes. CONCLUSIONS:Results suggest that the Mitf-Tbx2-Cyclin D1 pathway played an important role in regulation of melanocyte proliferation, and provided novel insights into the complex physiology of melanocytes.