Transcription factor (TF) DNA-binding dynamics govern cell fate and identity. However, our ability to pharmacologically control TF localization is limited. Here we leverage chemically driven binding site restriction leading to robust and DNA-sequence-specific redistribution of PU.
View Article and Find Full Text PDFTranscriptional dysregulation is a hallmark of diffuse large B cell lymphoma (DLBCL), as transcriptional regulators are frequently mutated. However, our mechanistic understanding of how normal transcriptional programs are co-opted in DLBCL has been hindered by a lack of methodologies that provide the temporal resolution required to separate direct and indirect effects on transcriptional control. We applied a chemical-genetic approach to engineer the inducible degradation of the transcription factor FOXO1, which is recurrently mutated (mFOXO1) in DLBCL.
View Article and Find Full Text PDFRecurrent chromosomal rearrangements found in rhabdomyosarcoma (RMS) produce the PAX3-FOXO1 fusion protein, which is an oncogenic driver and a dependency in this disease. One important function of PAX3-FOXO1 is to arrest myogenic differentiation, which is linked to the ability of RMS cells to gain an unlimited proliferation potential. Here, we developed a phenotypic screening strategy for identifying factors that collaborate with PAX3-FOXO1 to block myo-differentiation in RMS.
View Article and Find Full Text PDFCell type-specific gene expression is coordinated by DNA-encoded enhancers and the transcription factors (TFs) that bind to them in a sequence-specific manner. As such, these enhancers and TFs are critical mediators of normal development and altered enhancer or TF function is associated with the development of diseases such as cancer. While initially defined by their ability to activate gene transcription in reporter assays, putative enhancer elements are now frequently defined by their unique chromatin features including DNase hypersensitivity and transposase accessibility, bidirectional enhancer RNA (eRNA) transcription, CpG hypomethylation, high H3K27ac and H3K4me1, sequence-specific transcription factor binding, and co-factor recruitment.
View Article and Find Full Text PDFSomatic loss-of-function RUNX1 mutations in acute myeloid leukemia (AML) include missense, nonsense, and frameshift mutations, whereas germline RUNX1 variants in RUNX1-FPDMM also include large exonic deletions. Alternative variant detection approaches revealed that large exonic deletions in RUNX1 are also common in sporadic AML, which has implications for patient stratification and therapeutic decision-making. See related article by Eriksson et al.
View Article and Find Full Text PDFGenetic models suggested that SMARCA5 was required for DNA-templated events including transcription, DNA replication, and DNA repair. We engineered a degron tag into the endogenous alleles of SMARCA5, a catalytic component of the imitation switch complexes in three different human cell lines to define the effects of rapid degradation of this key regulator. Degradation of SMARCA5 was associated with a rapid increase in global nucleosome repeat length, which may allow greater chromatin compaction.
View Article and Find Full Text PDFTranscriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues, making it difficult to define the mechanism of transcription factor function using slow genetic methods. We used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO1, to define the mechanism by which it regulates gene expression programs. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses and integrating CUT&RUN, ATAC-seq, and eRNA analysis with deep proteomic analysis, we defined PAX3-FOXO1 function at a small network of direct transcriptional targets.
View Article and Find Full Text PDFAberrant epithelial differentiation and regeneration contribute to colon pathologies, including inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Myeloid translocation gene 16 (MTG16, also known as CBFA2T3) is a transcriptional corepressor expressed in the colonic epithelium. MTG16 deficiency in mice exacerbates colitis and increases tumor burden in CAC, though the underlying mechanisms remain unclear.
View Article and Find Full Text PDFBackground: All-trans retinoic acid (ATRA), a derivate of vitamin A, has been successfully used as a therapy to induce differentiation in M3 acute promyelocytic leukemia (APML), and has led to marked improvement in outcomes. Previously, attempts to use ATRA in non-APML in the clinic, however, have been underwhelming, likely due to persistent signaling through other oncogenic drivers. Dysregulated JAK/STAT signaling is known to drive several hematologic malignancies, and targeting JAK1 and JAK2 with the JAK1/JAK2 inhibitor ruxolitinib has led to improvement in survival in primary myelofibrosis and alleviation of vasomotor symptoms and splenomegaly in polycythemia vera and myelofibrosis.
View Article and Find Full Text PDFTranscriptional changes happen within minutes; however, RNAi or genetic deletion requires days to weeks before transcription networks can be analyzed. This limitation has made it challenging to distinguish direct from indirect targets of sequence-specific transcription factors. This inability to define direct transcriptional targets hinders detailed studies of transcriptional mechanisms.
View Article and Find Full Text PDFAccumulating evidence suggests that many immune responses are influenced by local nutrient concentrations in addition to the programming of intermediary metabolism within immune cells. Humoral immunity and germinal centers (GC) are settings in which these factors are under active investigation. Hypoxia is an example of how a particular nutrient is distributed in lymphoid follicles during an antibody response, and how oxygen sensors may impact the qualities of antibody output after immunization.
View Article and Find Full Text PDFTranscription factors regulate gene networks controlling normal hematopoiesis and are frequently deregulated in acute myeloid leukemia (AML). Critical to our understanding of the mechanism of cellular transformation by oncogenic transcription factors is the ability to define their direct gene targets. However, gene network cascades can change within minutes to hours, making it difficult to distinguish direct from secondary or compensatory transcriptional changes by traditional methodologies.
View Article and Find Full Text PDFPurpose: The BCL2 inhibitor, venetoclax, has transformed clinical care in acute myeloid leukemia (AML). However, subsets of patients do not respond or eventually acquire resistance. Venetoclax-based regimens can lead to considerable marrow suppression in some patients.
View Article and Find Full Text PDFHistone deacetylase 3 (Hdac3) is a target of the FDA approved HDAC inhibitors, which are used for the treatment of lymphoid malignancies. Here, we used Cd19-Cre to conditionally delete Hdac3 to define its role in germinal center B cells, which represent the cell of origin for many B cell malignancies. Cd19-Cre-Hdac3-/- mice showed impaired germinal center formation along with a defect in plasmablast production.
View Article and Find Full Text PDFIn the original version of this article the authors noted that the GEO accession number for the relevant dataset was listed incorrectly as GSE12454.
View Article and Find Full Text PDFThe t(8;21) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML). We found that t(8;21) AML were extremely sensitive to THZ1, which triggered apoptosis after only 4 h. We used precision nuclear run-on transcription sequencing (PROseq) to define the global effects of THZ1 and other CDK inhibitors on RNA polymerase II dynamics.
View Article and Find Full Text PDFInhibitors of the bromodomain and extraterminal domain family (BETi) offer a new approach to treat hematological malignancies, with leukemias containing mixed lineage leukemia rearrangements being especially sensitive due to a reliance on the regulation of transcription elongation. We explored the mechanism of action of BETi in cells expressing the t(8;21), and show that these compounds reduced the size of acute myeloid leukemia cells, triggered a rapid but reversible G /G arrest, and with time, cause cell death. Meta-analysis of PRO-seq data identified ribosomal genes, which are regulated by MYC, were downregulated within 3 hours of addition of the BETi.
View Article and Find Full Text PDFHistone deacetylase 3 (HDAC3) is the catalytic component of NCoR/SMRT corepressor complexes that mediate the actions of transcription factors implicated in the regulation of B-cell development and function. We crossed conditional knockout mice with knockin animals to delete in early progenitor B cells. The spleens of mice were virtually devoid of mature B cells, and B220CD43 B-cell progenitors accumulated within the bone marrow.
View Article and Find Full Text PDFNucleic Acids Res
July 2017
The genome-wide identification of microRNA transcription start sites (miRNA TSSs) is essential for understanding how miRNAs are regulated in development and disease. In this study, we developed mirSTP (mirna transcription Start sites Tracking Program), a probabilistic model for identifying active miRNA TSSs from nascent transcriptomes generated by global run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq). MirSTP takes advantage of characteristic bidirectional transcription signatures at active TSSs in GRO/PRO-seq data, and provides accurate TSS prediction for human intergenic miRNAs at a high resolution.
View Article and Find Full Text PDFUnlabelled: Somatic mutations in CREBBP occur frequently in B-cell lymphoma. Here, we show that loss of CREBBP facilitates the development of germinal center (GC)-derived lymphomas in mice. In both human and murine lymphomas, CREBBP loss-of-function resulted in focal depletion of enhancer H3K27 acetylation and aberrant transcriptional silencing of genes that regulate B-cell signaling and immune responses, including class II MHC.
View Article and Find Full Text PDFBromodomain and extra-terminal domain (BET) family inhibitors offer an approach to treating hematological malignancies. We used precision nuclear run-on transcription sequencing (PRO-seq) to create high-resolution maps of active RNA polymerases across the genome in t(8;21) acute myeloid leukemia (AML), as these polymerases are exceptionally sensitive to BET inhibitors. PRO-seq identified over 1,400 genes showing impaired release of promoter-proximal paused RNA polymerases, including the stem cell factor receptor tyrosine kinase KIT that is mutated in t(8;21) AML.
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