RESUMO
Attenuating aberrant transcriptional circuits holds great promise for the treatment of numerous diseases, including cancer. However, development of transcriptional inhibitors is hampered by the lack of a generally accepted functional cellular readout to characterize their target specificity and on-target activity. We benchmarked the direct gene-regulatory signatures of six agents reported as inhibitors of the oncogenic transcription factor MYB against targeted MYB degradation in a nascent transcriptomics assay. The inhibitors demonstrated partial specificity for MYB target genes but displayed significant off-target activity. Unexpectedly, the inhibitors displayed bimodal on-target effects, acting as mixed agonists-antagonists. Our data uncover unforeseen agonist effects of small molecules originally developed as TF inhibitors and argue that rapid-kinetics benchmarking against degron models should be used for functional characterization of transcriptional modulators.
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rRNAs are transcribed from ribosomal DNA (rDNA) repeats, the most intensively transcribed loci in the genome. Due to their repetitive nature, there is a lack of genome assemblies suitable for rDNA mapping, creating a vacuum in our understanding of how the most abundant RNA in the cell is regulated. Our recent work revealed binding of numerous mammalian transcription and chromatin factors to rDNA. Several of these factors were known to play critical roles in development, tissue function, and malignancy, but their potential roles in rDNA regulation remained unexplored. This demonstrated the blind spot into which rDNA has fallen in genetic and epigenetic studies and highlighted an unmet need for public rDNA-optimized genome assemblies. Here, we customized five human and mouse assemblies-hg19 (GRCh37), hg38 (GRCh38), hs1 (T2T-CHM13), mm10 (GRCm38), and mm39 (GRCm39)-to render them suitable for rDNA mapping. The standard builds of these genomes contain numerous fragmented or repetitive rDNA loci. We identified and masked all rDNA-like regions, added a single rDNA reference sequence of the appropriate species as a â¼45 kb chromosome designated "chromosome R," and created annotation files to aid visualization of rDNA features in browser tracks. We validated these customized genomes for mapping of known rDNA-binding proteins and present a simple workflow for mapping chromatin immunoprecipitation-sequencing datasets. Customized genome assemblies, annotation files, positive and negative control tracks, and Snapgene files of standard rDNA reference sequences have been deposited to GitHub. These resources make rDNA mapping and visualization more readily accessible to a broad audience.
Assuntos
DNA Ribossômico , Genoma , Animais , Humanos , Camundongos , Cromatina , Mapeamento Cromossômico , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Mamíferos/metabolismo , RNA RibossômicoRESUMO
Lineage-defining transcription factors form densely interconnected circuits in chromatin occupancy assays, but the functional significance of these networks remains underexplored. We reconstructed the functional topology of a leukemia cell transcription network from the direct gene-regulatory programs of eight core transcriptional regulators established in pre-steady state assays coupling targeted protein degradation with nascent transcriptomics. The core regulators displayed narrow, largely non-overlapping direct transcriptional programs, forming a sparsely interconnected functional hierarchy stabilized by incoherent feed-forward loops. BET bromodomain and CDK7 inhibitors disrupted the core regulators' direct programs, acting as mixed agonists/antagonists. The network is predictive of dynamic gene expression behaviors in time-resolved assays and clinically relevant pathway activity in patient populations.
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Ribosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2,200 ChIP-seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA and identified robust binding of multiple TF families to canonical TF motifs on rDNA. Using a 47S-FISH-Flow assay developed for nascent rRNA quantification, we demonstrated that targeted degradation of C/EBP alpha (CEBPA), a critical hematopoietic TF with conserved rDNA binding, caused rapid reduction in rRNA transcription due to reduced RNA Pol I occupancy. Our work identifies numerous potential rRNA regulators and provides a template for dissection of TF roles in rRNA transcription.
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RNA Polimerase I , Fatores de Transcrição , Humanos , Camundongos , Animais , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , RNA Ribossômico/genética , Transcrição Gênica , DNA Ribossômico/genética , RNA , CromatinaRESUMO
Relapse of acute myeloid leukemia (AML) after allogeneic bone marrow transplantation has been linked to immune evasion due to reduced expression of major histocompatibility complex class II (MHCII) genes through unknown mechanisms. In this work, we developed CORENODE, a computational algorithm for genome-wide transcription network decomposition that identified a transcription factor (TF) tetrad consisting of IRF8, MYB, MEF2C, and MEIS1, regulating MHCII expression in AML cells. We show that reduced MHCII expression at relapse is transcriptionally driven by combinatorial changes in the expression of these TFs, where MYB and IRF8 play major opposing roles, acting independently of the IFNγ/CIITA pathway. Beyond the MHCII genes, MYB and IRF8 antagonistically regulate a broad genetic program responsible for cytokine signaling and T-cell stimulation that displays reduced expression at relapse. A small number of cells with altered TF abundance and silenced MHCII expression are present at the time of initial leukemia diagnosis, likely contributing to eventual relapse. SIGNIFICANCE: Our findings point to an adaptive transcriptional mechanism of AML evolution after allogeneic transplantation whereby combinatorial fluctuations of TF expression under immune pressure result in the selection of cells with a silenced T-cell stimulation program. This article is highlighted in the In This Issue feature, p. 369.
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Leucemia Mieloide Aguda , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Fatores Reguladores de Interferon , Leucemia Mieloide Aguda/genética , Recidiva , Transplante HomólogoRESUMO
High-risk forms of B-acute lymphoblastic leukemia (B-ALL) remain a therapeutic challenge. Leukemia-initiating cells (LICs) self-renew and spark relapse and therefore have been the subject of intensive investigation; however, the properties of LICs in high-risk B-ALL are not well understood. Here, we use single-cell transcriptomics and quantitative xenotransplantation to understand LICs in MLL-rearranged (MLL-r) B-ALL. Compared with reported LIC frequencies in acute myeloid leukemia (AML), engraftable LICs in MLL-r B-ALL are abundant. Although we find that multipotent, self-renewing LICs are enriched among phenotypically undifferentiated B-ALL cells, LICs with the capacity to replenish the leukemic cellular diversity can emerge from more mature fractions. While inhibiting oxidative phosphorylation blunts blast proliferation, this intervention promotes LIC emergence. Conversely, inhibiting hypoxia and glycolysis impairs MLL-r B-ALL LICs, providing a therapeutic benefit in xenotransplantation systems. These findings provide insight into the aggressive nature of MLL-r B-ALL and provide a rationale for therapeutic targeting of hypoxia and glycolysis.
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Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células Precursoras , Glicólise , Humanos , Hipóxia , Leucemia Mieloide Aguda/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMO
Leukemic blasts are immune cells gone awry. We hypothesized that dysregulation of inflammatory pathways contributes to the maintenance of their leukemic state and can be exploited as cell-intrinsic, self-directed immunotherapy. To this end, we applied genome-wide screens to discover genetic vulnerabilities in acute myeloid leukemia (AML) cells implicated in inflammatory pathways. We identified the immune modulator IRF2BP2 as a selective AML dependency. We validated AML cell dependency on IRF2BP2 with genetic and protein degradation approaches in vitro and genetically in vivo. Chromatin and global gene-expression studies demonstrated that IRF2BP2 represses IL1ß/TNFα signaling via NFκB, and IRF2BP2 perturbation results in an acute inflammatory state leading to AML cell death. These findings elucidate a hitherto unexplored AML dependency, reveal cell-intrinsic inflammatory signaling as a mechanism priming leukemic blasts for regulated cell death, and establish IRF2BP2-mediated transcriptional repression as a mechanism for blast survival. SIGNIFICANCE: This study exploits inflammatory programs inherent to AML blasts to identify genetic vulnerabilities in this disease. In doing so, we determined that AML cells are dependent on the transcriptional repressive activity of IRF2BP2 for their survival, revealing cell-intrinsic inflammation as a mechanism priming leukemic blasts for regulated cell death. See related commentary by Puissant and Medyouf, p. 1617. This article is highlighted in the In This Issue feature, p. 1599.
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Leucemia Mieloide Aguda , Humanos , Inflamação/genética , Leucemia Mieloide Aguda/genética , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
Acute myeloid leukemia with KMT2A (MLL) rearrangements is characterized by specific patterns of gene expression and enhancer architecture, implying unique core transcriptional regulatory circuitry. Here, we identified the transcription factors MEF2D and IRF8 as selective transcriptional dependencies of KMT2A-rearranged AML, where MEF2D displays partially redundant functions with its paralog, MEF2C. Rapid transcription factor degradation followed by measurements of genome-wide transcription rates and superresolution microscopy revealed that MEF2D and IRF8 form a distinct core regulatory module with a narrow direct transcriptional program that includes activation of the key oncogenes MYC, HOXA9, and BCL2. Our study illustrates a mechanism of context-specific transcriptional addiction whereby a specific AML subclass depends on a highly specialized core regulatory module to directly enforce expression of common leukemia oncogenes.
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Leucemia Mieloide Aguda , Proteína de Leucina Linfoide-Mieloide , Rearranjo Gênico , Humanos , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Leucemia Mieloide Aguda/genética , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Oncogenes/genéticaRESUMO
We used mouse ENCODE data along with complementary data from other laboratories to study the dynamics of occupancy and the role in gene regulation of the transcription factor TAL1, a critical regulator of hematopoiesis, at multiple stages of hematopoietic differentiation. We combined ChIP-seq and RNA-seq data in six mouse cell types representing a progression from multilineage precursors to differentiated erythroblasts and megakaryocytes. We found that sites of occupancy shift dramatically during commitment to the erythroid lineage, vary further during terminal maturation, and are strongly associated with changes in gene expression. In multilineage progenitors, the likely target genes are enriched for hematopoietic growth and functions associated with the mature cells of specific daughter lineages (such as megakaryocytes). In contrast, target genes in erythroblasts are specifically enriched for red cell functions. Furthermore, shifts in TAL1 occupancy during erythroid differentiation are associated with gene repression (dissociation) and induction (co-occupancy with GATA1). Based on both enrichment for transcription factor binding site motifs and co-occupancy determined by ChIP-seq, recruitment by GATA transcription factors appears to be a stronger determinant of TAL1 binding to chromatin than the canonical E-box binding site motif. Studies of additional proteins lead to the model that TAL1 regulates expression after being directed to a distinct subset of genomic binding sites in each cell type via its association with different complexes containing master regulators such as GATA2, ERG, and RUNX1 in multilineage cells and the lineage-specific master regulator GATA1 in erythroblasts.
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Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição GATA/metabolismo , Regulação da Expressão Gênica , Hematopoese , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sítios de Ligação , Diferenciação Celular/genética , Cromatina/genética , Cromatina/metabolismo , Imunoprecipitação da Cromatina , Análise por Conglomerados , Biologia Computacional , Conjuntos de Dados como Assunto , Células Eritroides/citologia , Células Eritroides/metabolismo , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Histonas/metabolismo , Camundongos , Modelos Biológicos , Anotação de Sequência Molecular , Motivos de Nucleotídeos , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Proteína 1 de Leucemia Linfocítica Aguda de Células T , TranscriptomaRESUMO
Combinatorial actions of relatively few transcription factors control hematopoietic differentiation. To investigate this process in erythro-megakaryopoiesis, we correlated the genome-wide chromatin occupancy signatures of four master hematopoietic transcription factors (GATA1, GATA2, TAL1, and FLI1) and three diagnostic histone modification marks with the gene expression changes that occur during development of primary cultured megakaryocytes (MEG) and primary erythroblasts (ERY) from murine fetal liver hematopoietic stem/progenitor cells. We identified a robust, genome-wide mechanism of MEG-specific lineage priming by a previously described stem/progenitor cell-expressed transcription factor heptad (GATA2, LYL1, TAL1, FLI1, ERG, RUNX1, LMO2) binding to MEG-associated cis-regulatory modules (CRMs) in multipotential progenitors. This is followed by genome-wide GATA factor switching that mediates further induction of MEG-specific genes following lineage commitment. Interaction between GATA and ETS factors appears to be a key determinant of these processes. In contrast, ERY-specific lineage priming is biased toward GATA2-independent mechanisms. In addition to its role in MEG lineage priming, GATA2 plays an extensive role in late megakaryopoiesis as a transcriptional repressor at loci defined by a specific DNA signature. Our findings reveal important new insights into how ERY and MEG lineages arise from a common bipotential progenitor via overlapping and divergent functions of shared hematopoietic transcription factors.
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Diferenciação Celular , Linhagem da Célula , Eritropoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Trombopoese/fisiologia , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Análise por Conglomerados , Fator de Transcrição GATA1/metabolismo , Fator de Transcrição GATA2/metabolismo , Perfilação da Expressão Gênica , Inativação Gênica , Estudo de Associação Genômica Ampla , Histonas/metabolismo , Camundongos , Modelos Biológicos , Motivos de Nucleotídeos , Ligação Proteica , Proteína Proto-Oncogênica c-fli-1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteína 1 de Leucemia Linfocítica Aguda de Células T , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The calcium regulated calcineurin-nuclear factor of activated T cells (NFAT) pathway modulates the physiology of numerous cell types, including hematopoietic. Upon activation, calcineurin dephosphorylates NFAT family transcription factors, triggering their nuclear entry and activation or repression of target genes. NFATc1 and c2 isoforms are expressed in megakaryocytes. Moreover, human chromosome 21 (Hsa21) encodes several negative regulators of calcineurin-NFAT, candidates in the pathogenesis of Down syndrome (trisomy 21)-associated transient myeloproliferative disorder and acute megakaryoblastic leukemia. To investigate the role of calcineurin-NFAT in megakaryopoiesis, we examined wild-type mice treated with the calcineurin inhibitor cyclosporin A and transgenic mice expressing a targeted single extra copy of Dscr1, an Hsa21-encoded calcineurin inhibitor. Both murine models exhibited thrombocytosis with increased megakaryocytes and megakaryocyte progenitors. Pharmacological or genetic inhibition of calcineurin in mice caused accumulation of megakaryocytes exhibiting enhanced 5-bromo-2'-deoxyuridine uptake and increased expression of messenger RNAs encoding CDK4 and G1 cyclins, which promote cell division. Additionally, human megakaryocytes with trisomy 21 show increased proliferation and decreased NFAT activation compared with euploid controls. Our data indicate that inhibition of calcineurin-NFAT drives proliferation of megakaryocyte precursors by de-repressing genes that drive cell division, providing insights into mechanisms of normal megakaryopoiesis and megakaryocytic abnormalities that accompany Down syndrome.
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Calcineurina/metabolismo , Células Progenitoras de Megacariócitos/citologia , Megacariócitos/citologia , Fatores de Transcrição NFATC/metabolismo , Trombopoese , Animais , Apoptose , Proteínas de Ligação ao Cálcio , Ciclo Celular , Proliferação de Células , Células Cultivadas , Síndrome de Down/metabolismo , Proteína Ligante Fas/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células Progenitoras de Megacariócitos/metabolismo , Megacariócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Musculares/genética , Contagem de Plaquetas , Transdução de SinaisRESUMO
The Bateman domain (CBS subdomain) of IMP dehydrogenase (IMPDH), a rate-limiting enzyme of the de novo GMP biosynthesis, is evolutionarily conserved but has no established function. Deletion of the Bateman domain has no effect on the in vitro IMPDH activity. We report that in vivo deletion of the Bateman domain of IMPDH in Escherichia coli (guaB(DeltaCBS)) sensitizes the bacterium to growth arrest by adenosine and inosine. These nucleosides exert their growth inhibitory effect via a dramatic increase in the intracellular adenylate nucleotide pool, which results in the enhanced allosteric inhibition of PRPP synthetase and consequently a PRPP deficit. The ensuing starvation for pyrimidine nucleotides culminates in growth arrest. Thus, deletion of the Bateman domain of IMPDH derepresses the synthesis of AMP from IMP. The growth inhibitory effect of inosine can be rescued by second-site suppressor mutations in the genes responsible for the conversion of inosine to AMP (gsk, purA, and purB) as well as by the prsA1 allele, which encodes a PRPP synthetase that is insensitive to allosteric inhibition by adenylate nucleotides. Importantly, the guaB(DeltaCBS) phenotype can be complemented in trans by a mutant guaB allele, which encodes a catalytically disabled IMPDH(C305A) protein containing an intact Bateman domain. We conclude that the Bateman domain of IMPDH is a negative trans-regulator of adenylate nucleotide synthesis, and that this role is independent of the catalytic function of IMPDH in the de novo GMP biosynthesis.
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Adenosina/biossíntese , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Guanosina Monofosfato/biossíntese , IMP Desidrogenase/metabolismo , Adenosina/genética , Catálise , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , IMP Desidrogenase/genética , Inosina/genética , Inosina/metabolismo , Mutação , Estrutura Terciária de ProteínaRESUMO
Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyses the rate-limiting step in guanine nucleotide biosynthesis. IMPDH has an evolutionary conserved CBS subdomain of unknown function. The subdomain can be deleted without impairing the in vitro IMPDH catalytic activity and is the site for mutations associated with human retinitis pigmentosa. A guanine-prototrophic Escherichia coli strain, MP101, was constructed with the subdomain sequence deleted from the chromosomal gene for IMPDH. The ATP content was substantially elevated in MP101 whereas the GTP content was slighty reduced. The activities of IMPDH, adenylosuccinate synthetase and GMP reductase were two to threefold lower in MP101 crude extracts compared with the BW25113 wild-type strain. Guanine induced a threefold reduction in the MP101 ATP pool and a fourfold increase in the GTP pool within 10 min of addition to growing cells; this response does not result from the reduced IMPDH activity or starvation for guanylates. In vivo kinetic analysis using 14-C tracers and 33-P pulse-chasing revealed mutation-associated changes in purine nucleotide fluxes and turnover rates. We conclude that the CBS subdomain of IMPDH may coordinate the activities of the enzymes of purine nucleotide metabolism and is essential for maintaining the normal ATP and GTP pool sizes in E. coli.
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Escherichia coli/enzimologia , IMP Desidrogenase/metabolismo , Nucleotídeos de Purina/metabolismo , Trifosfato de Adenosina/análise , Adenilossuccinato Sintase/metabolismo , Radioisótopos de Carbono/metabolismo , Escherichia coli/química , Escherichia coli/genética , GMP Redutase/metabolismo , Guanina/metabolismo , Guanosina Trifosfato/análise , IMP Desidrogenase/química , IMP Desidrogenase/genética , Modelos Biológicos , Modelos Moleculares , Mutação , Radioisótopos de Fósforo/metabolismo , Estrutura Terciária de Proteína , Deleção de Sequência , Streptococcus pyogenes/enzimologiaRESUMO
BACKGROUND: The detection of unknown mutations is important in research and medicine. For this purpose, a mismatch-specific endonuclease CEL I from celery has been established as a useful tool in high throughput projects. Previously, CEL I-like activities were described only in a variety of plants and could not be expressed in an active form in bacteria. RESULTS: We describe expression of active recombinant plant mismatch endonucleases and modification of their activities. We also report the cloning of a CEL I ortholog from Spinacia oleracea (spinach) which we termed SP I nuclease. Active CEL I and SP I nucleases were expressed as C-terminal hexahistidine fusions and affinity purified from the cell culture media. Both recombinant enzymes were active in mutation detection in BRCA1 gene of patient-derived DNA. Native SP nuclease purified from spinach is unable to incise at single-nucleotide substitutions and loops containing a guanine nucleotide, but the recombinant SP I nuclease can cut at these sites. CONCLUSION: The insect cell-expressed CEL I orthologs may not be identical to their native counterparts purified from plant tissues. The present expression system should facilitate further development of CEL I-based mutation detection technologies.