FUS Oncofusion Protein Condensates Recruit mSWI/SNF Chromatin Remodelers via Heterotypic Interactions Between Prion-like Domains

AbstractFusion transcription factors generated by genomic translocations are common drivers of several types of cancers including sarcomas and leukemias. Oncofusions of the FET (FUS, EWSR1, and TAF15) family of proteins result from fusion of the prion-like domain (PLD) of FET proteins to the DNA-binding domain (DBD) of certain transcription regulators and are implicated in aberrant transcriptional programs through interactions with chromatin remodelers. Here, we show that FUS-DDIT3, a FET oncofusion protein, undergoes PLD-mediated phase separation into liquid-like condensates. Nuclear FUS-DDIT3 condensates can recruit essential components of the global transcriptional machinery such as the chromatin remodeler SWI/SNF. The recruitment of mammalian SWI/SNF is driven by heterotypic PLD-PLD interactions between FUS-DDIT3 and core subunits of SWI/SNF, such as the catalytic component BRG1. Further experiments with single-molecule correlative force-fluorescence microscopy support a model wherein the fusion protein forms condensates on DNA surface and enrich BRG1 to activate transcription by ectopic chromatin remodeling. Similar PLD-driven co-condensation of mSWI/SNF with transcription factors can be employed by other oncogenic fusion proteins with a generic PLD-DBD domain architecture for global transcriptional reprogramming.

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Single-molecule dynamics and genome-wide transcriptomics reveal that NF-κB (p65)-DNA binding times can be decoupled from transcriptional activation

10.1101/255380 ◽

2018 ◽

Author(s):

Andrea Callegari ◽

Christian Sieben ◽

Alexander Benke ◽

David M. Suter ◽

Beat Fierz ◽

...

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Single Molecule ◽

Protein Interaction ◽

Transcriptional Activation ◽

Dna Binding Domain ◽

Protein Protein Interaction ◽

Genome Wide ◽

Binding Time ◽

Transcriptional Output

AbstractTranscription factors (TFs) regulate gene expression in both prokaryotes and eukaryotes by recognizing and binding to specific DNA promoter sequences. In higher eukaryotes, it remains unclear how the duration of TF binding to DNA relates to downstream transcriptional output. Here, we address this question for the transcriptional activator NF-κB (p65), by live-cell single molecule imaging of TF-DNA binding kinetics and genome-wide quantification of p65-mediated transcription. We used mutants of p65, perturbing either the DNA binding domain (DBD) or the protein-protein transactivation domain (TAD). We found that p65-DNA binding time was predominantly determined by its DBD and directly correlated with its transcriptional output as long as the TAD is intact. Surprisingly, mutation or deletion of the TAD did not modify p65-DNA binding stability, suggesting that the p65 TAD generally contributes neither to the assembly of an “enhanceosome,” nor to the active removal of p65 from putative specific binding sites. However, TAD removal did reduce p65-mediated transcriptional activation, indicating that protein-protein interactions act to translate the long-lived p65-DNA binding into productive transcription.Author SummaryTo control transcription of a certain gene or a group of genes, both eukaryotes and prokaryotes express specialized proteins, transcription factors (TFs). During gene activation, TFs bind gene promotor sequences to recruit the transcriptional machinery including DNA polymerase II. TFs are often multi-subunit proteins containing a DNA-binding domain (DBD) as well as a protein-protein interaction interface. It was suggested that the duration of a TF-DNA binding event 1) depends on these two subunits and 2) dictates the outcome, i.e. the amount of mRNA produced from an activated gene. We set out to address these hypotheses using the transcriptional activator NF-κB (p65) as well as a number of mutants affecting different functional subunits. Using a combination of live-cell microscopy and RNA sequencing, we show that p65 DNA-binding time indeed correlates with the transcriptional output, but that this relationship depends on, and hence can be uncoupled by altering, the protein-protein interaction capacity. Our results suggest that, while p65 DNA binding times are dominated by the DBD, a transcriptional output can only be achieved with a functional protein-protein interaction subunit.

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ATP hydrolysis coordinates the activities of two motors in a dimeric chromatin remodeling enzyme

10.1101/2020.11.17.387662 ◽

2020 ◽

Author(s):

Stephanie L. Johnson ◽

Geeta Narlikar

Keyword(s):

Chromatin Remodeling ◽

Single Molecule ◽

Molecular Motors ◽

Atp Hydrolysis ◽

Oligomeric State ◽

Chromatin Remodelers ◽

Chromatin Dynamics ◽

A Cell ◽

Conventional Methods ◽

Eukaryotic Genomes

AbstractATP-dependent chromatin remodelers are essential enzymes that restructure eukaryotic genomes to enable all DNA-based processes. The diversity and complexity of these processes are matched by the complexity of the enzymes that carry them out, making remodelers a challenging class of molecular motors to study by conventional methods. Here we use a single molecule biophysical assay to overcome some of these challenges, enabling a detailed mechanistic dissection of a paradigmatic remodeler reaction, that of sliding a nucleosome towards the longer DNA linker. We focus on how two motors of a dimeric remodeler coordinate to accomplish such directional sliding. We find that ATP hydrolysis by both motors promotes coordination, suggesting a role for ATP in resolving the competition for directional commitment. Furthermore, we show an artificially constitutive dimer is no more or less coordinated, but is more processive, suggesting a cell could modulate a remodeler’s oligomeric state to modulate local chromatin dynamics.

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Histone Deacetylase Inhibitors Induce FPGS mRNA Expression and Intracellular Accumulation of Long-Chain Methotrexate Polyglutamates in Childhood Acute Lymphoblastic Leukemia: Implications for Combination Therapy

Blood ◽

10.1182/blood.v112.11.2923.2923 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2923-2923

Author(s):

Guy J Leclerc ◽

Caihong Mou ◽

Gilles M Leclerc ◽

Abdul M Mian ◽

Julio C Barredo

Keyword(s):

Transcription Factors ◽

Mrna Expression ◽

Histone Deacetylase ◽

Cell Lines ◽

Chromatin Remodeling ◽

Histone Deacetylase Inhibitors ◽

Lymphoblastic Leukemia ◽

Intracellular Accumulation ◽

Essential Components ◽

Long Chain

Abstract Folate cofactors are essential components of one carbon metabolism and are required for the biosynthesis of purines, pyrimidines, serine and methionine. The classical folate antagonist methotrexate (MTX) continues to be a universal component of most ALL treatment regimens. MTX is retained within cells as long-chain polyglutamates (MTX-PGs) after metabolism by the enzyme folylpoly-γ -glutamate synthetase (FPGS). Intracellular retention of MTX-PGs results in enhanced cytotoxicity due to prolonged inhibition of dihydrofolate reductase (DHFR) and thymidylate synthetase (TS). The FPGS gene is regulated by the transcription factors NFY and Sp1. Using DNaseI assays we identified a hypersensitive site mapping closely upstream of exon 1, suggesting that chromatin remodeling may contribute to FPGS gene regulation. To investigate the role of histone modifications and chromatin remodeling on FPGS expression and uncover interactions between NFY, Sp1 and HDAC1, we performed co-immunoprecipitation and Western blotting. Our results demonstrate that HDAC1 complexes with NFY and Sp1 transcription factors in both B- and T-ALL cells. DNA affinity precipitation assays (DAPA) revealed that HDAC1 is recruited by NFY and Sp1 to the FPGS promoter. These findings suggest that transcription of the FPGS gene may be regulated by NFY and Sp1 factors interacting with HDAC1, and leading to chromatin remodeling. We then examined the effect of the histone deacetylase inhibitors (HDACIs) sodium butyrate (NaBu) and suberoylanilide hydroxamic acid (SAHA) on the expression of FPGS and other folate-related genes in NALM6 (Bp-ALL), REH (TEL/AML1+, Bp-ALL), SupB15 (BCR/ABL+, Bp-ALL), and CCRF-CEM (T-ALL) cells using qRT-PCR. In all cell lines examined, treatment with HDACIs increased FPGS mRNA expression by 2- to 5-fold, whereas the level of DHFR and TS mRNA expression were decreased. On this basis, we hypothesized that induction of FPGS expression by HDACIs, results in higher accumulation of MTX-PG and enhanced MTX cytotoxicity in ALL cells. Further, the concomitant decrease in the expression of the MTX-PG target enzymes DHFR and TS, would enhance the cytotoxicity of the combination of HDACIs plus MTX in ALL cells. To test this hypothesis, NALM6, REH, and SupB15 cells were treated with MTX (4h) + SAHA (24h), and cell viability assessed. We determined that SAHA increased the intracellular accumulation of long chain MTX-PGs (n ≥3 Glu) in ALL cells, correlating with the upregulation of FPGS expression in SAHA-treated cells. Treatment with MTX + SAHA increased cytotoxicity by ~30% with a calculated combination index of ≤ 0.8 indicating synergy. Analysis of apoptosis using AnnexinV/PI staining revealed a 2 to 3-fold increase in apoptotic cell death in all cell lines treated with this combination. Our data suggest HDACIs enhance MTX cytotoxicity by upregulation of FPGS expression, increased accumulation of MTXPG and downregulation of DHFR and TS. The synergism exhibited by the combination of MTX and SAHA suggests it should be tested in ALL patients, in particular those who exhibit phenotypes with de novo or acquired resistance to MTX.

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Actin-related protein 5 functions as a novel modulator of MyoD and MyoG in skeletal muscle and in rhabdomyosarcoma

10.1101/2021.05.27.446008 ◽

2021 ◽

Author(s):

Tsuyoshi Morita ◽

Ken'ichiro Hayashi

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Transcription Factors ◽

Chromatin Remodeling ◽

Myogenic Differentiation ◽

Related Protein ◽

Chromatin Remodelers ◽

Hind Limbs ◽

Muscle Tissues ◽

Extension Class

Myogenic regulatory factors (MRFs) are pivotal transcription factors in myogenic differentiation. MyoD commits cells to the skeletal muscle lineage by inducing myogenic genes through recruitment of chromatin remodelers to its target loci. This study showed that Actin-related protein 5 (Arp5) acts as an inhibitory regulator of MyoD and MyoG by binding to their cysteine-rich (CR) region, which overlaps with the region essential for their epigenetic functions. Arp5 expression was faint in skeletal muscle tissues. Excessive Arp5 in mouse hind limbs caused skeletal muscle fiber atrophy. Further, Arp5 overexpression in myoblasts inhibited myotube formation by diminishing myogenic gene expression, whereas Arp5 depletion augmented myogenic gene expression. Arp5 disturbed MyoD-mediated chromatin remodeling through competition with the three-amino-acid-loop-extension-class homeodomain transcription factors the Pbx1–Meis1 heterodimer for binding to the CR region. This antimyogenic function was independent of the INO80 chromatin remodeling complex, although Arp5 is an important component of that. In rhabdomyosarcoma (RMS) cells, Arp5 expression was significantly higher than in normal myoblasts and skeletal muscle tissue, probably contributing to MyoD and MyoG activity dysregulation. Arp5 depletion in RMS partially restored myogenic properties while inhibiting tumorigenic properties. Thus, Arp5 is a novel modulator of MRFs in skeletal muscle differentiation.

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Single-fiber nucleosome density shapes the regulatory output of a mammalian chromatin remodeling enzyme

10.1101/2021.12.10.472156 ◽

2021 ◽

Author(s):

Nour J Abdulhay ◽

Laura J Hsieh ◽

Colin P McNally ◽

Mythili Ketavarapu ◽

Sivakanthan Kasinathan ◽

...

Keyword(s):

Chromatin Remodeling ◽

Single Molecule ◽

Single Fiber ◽

Chromatin Remodelers ◽

Linker Length ◽

Dna Accessibility ◽

Nucleosome Density ◽

Regulatory Effects

ABSTRACTATP-dependent chromatin remodelers regulate the DNA accessibility required of virtually all nuclear processes. Biochemical studies have provided insight into remodeler action at the nucleosome level, but how these findings translate to activity on chromatin fibers in vitro and in vivo remains poorly understood. Here, we present a massively multiplex single-molecule platform allowing high-resolution mapping of nucleosomes on fibers assembled on mammalian genomic sequences. We apply this method to distinguish between competing models for chromatin remodeling by the essential ISWI ATPase SNF2h: linker-length-dependent dynamic positioning versus fixed-linker-length static clamping. Our single-fiber data demonstrate that SNF2h operates as a density-dependent, length-sensing chromatin remodeler whose ability to decrease or increase DNA accessibility depends on single-fiber nucleosome density. In vivo, this activity manifests as different regulatory modes across epigenomic domains: at canonically-defined heterochromatin, SNF2h generates evenly-spaced nucleosome arrays of multiple nucleosome repeat lengths; at SNF2h-dependent accessible sites, SNF2h slides nucleosomes to increase accessibility of motifs for the essential transcription factor CTCF. Overall, our generalizable approach provides molecularly-precise views of the processes that shape nuclear physiology. Concurrently, our data illustrate how a mammalian chromatin remodeling enzyme can effectively sense nucleosome density to induce diametrically-opposed regulatory effects within the nucleus.

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Deciphering the enigma of missing DNA binding domain of LacI family transcription factors

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2021.109060 ◽

2021 ◽

Vol 713 ◽

pp. 109060

Author(s):

Neetu Neetu ◽

Madhusudhanarao Katiki ◽

Jai Krishna Mahto ◽

Monica Sharma ◽

Anoop Narayanan ◽

...

Keyword(s):

Transcription Factors ◽

Dna Binding ◽

Binding Domain ◽

Dna Binding Domain ◽

Laci Family

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The trithorax group gene osa encodes an ARID-domain protein that genetically interacts with the brahma chromatin-remodeling factor to regulate transcription

Development ◽

10.1242/dev.126.4.733 ◽

1999 ◽

Vol 126 (4) ◽

pp. 733-742 ◽

Cited By ~ 1

Author(s):

M. Vazquez ◽

L. Moore ◽

J.A. Kennison

Keyword(s):

Gene Regulation ◽

Chromatin Remodeling ◽

Homeotic Gene ◽

Dna Binding Domain ◽

Atpase Subunit ◽

Trithorax Group ◽

Chromatin Remodeling Complex ◽

P2 Promoter ◽

Domain Protein ◽

Arid Domain

The trithorax group gene brahma (brm) encodes the ATPase subunit of a chromatin-remodeling complex involved in homeotic gene regulation. We report here that brm interacts with another trithorax group gene, osa, to regulate the expression of the Antennapedia P2 promoter. Regulation of Antennapedia by BRM and OSA proteins requires sequences 5′ to the P2 promoter. Loss of maternal osa function causes severe segmentation defects, indicating that the function of osa is not limited to homeotic gene regulation. The OSA protein contains an ARID domain, a DNA-binding domain also present in the yeast SWI1 and Drosophila DRI proteins. We propose that the OSA protein may target the BRM complex to Antennapedia and other regulated genes.

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Reduction in gene expression noise by targeted increase in accessibility at gene loci

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2018640118 ◽

2021 ◽

Vol 118 (42) ◽

pp. e2018640118

Author(s):

LaTasha C. R. Fraser ◽

Ryan J. Dikdan ◽

Supravat Dey ◽

Abhyudai Singh ◽

Sanjay Tyagi

Keyword(s):

Gene Expression ◽

Transcription Factors ◽

Single Molecule ◽

Single Cells ◽

Global Changes ◽

Messenger Rnas ◽

Histone Acetyl Transferase ◽

Gene Expression Noise ◽

Expression Noise ◽

Eukaryotic Genes

Many eukaryotic genes are expressed in randomly initiated bursts that are punctuated by periods of quiescence. Here, we show that the intermittent access of the promoters to transcription factors through relatively impervious chromatin contributes to this “noisy” transcription. We tethered a nuclease-deficient Cas9 fused to a histone acetyl transferase at the promoters of two endogenous genes in HeLa cells. An assay for transposase-accessible chromatin using sequencing showed that the activity of the histone acetyl transferase altered the chromatin architecture locally without introducing global changes in the nucleus and rendered the targeted promoters constitutively accessible. We measured the gene expression variability from the gene loci by performing single-molecule fluorescence in situ hybridization against mature messenger RNAs (mRNAs) and by imaging nascent mRNA molecules present at active gene loci in single cells. Because of the increased accessibility of the promoter to transcription factors, the transcription from two genes became less noisy, even when the average levels of expression did not change. In addition to providing evidence for chromatin accessibility as a determinant of the noise in gene expression, our study offers a mechanism for controlling gene expression noise which is otherwise unavoidable.

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The Long Road to Understanding RNAPII Transcription Initiation and Related Syndromes

Annual Review of Biochemistry ◽

10.1146/annurev-biochem-090220-112253 ◽

2021 ◽

Vol 90 (1) ◽

pp. 193-219

Author(s):

Emmanuel Compe ◽

Jean-Marc Egly

Keyword(s):

Transcription Factors ◽

Rna Polymerase Ii ◽

Chromatin Remodeling ◽

Transcription Initiation ◽

Rna Synthesis ◽

Regulatory Elements ◽

Initiation Mechanism ◽

Protein Coding ◽

Core Promoters ◽

General Transcription Factors

In eukaryotes, transcription of protein-coding genes requires the assembly at core promoters of a large preinitiation machinery containing RNA polymerase II (RNAPII) and general transcription factors (GTFs). Transcription is potentiated by regulatory elements called enhancers, which are recognized by specific DNA-binding transcription factors that recruit cofactors and convey, following chromatin remodeling, the activating cues to the preinitiation complex. This review summarizes nearly five decades of work on transcription initiation by describing the sequential recruitment of diverse molecular players including the GTFs, the Mediator complex, and DNA repair factors that support RNAPII to enable RNA synthesis. The elucidation of the transcription initiation mechanism has greatly benefited from the study of altered transcription components associated with human diseases that could be considered transcription syndromes.

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CDK8/19 Mediator kinases potentiate induction of transcription by NFκB

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1710467114 ◽

2017 ◽

Vol 114 (38) ◽

pp. 10208-10213 ◽

Cited By ~ 35

Author(s):

Mengqian Chen ◽

Jiaxin Liang ◽

Hao Ji ◽

Zhengguan Yang ◽

Serena Altilia ◽

...

Keyword(s):

Transcription Factors ◽

Rna Polymerase Ii ◽

Kinase Inhibitors ◽

Viral Infections ◽

Nuclear Factor Κb ◽

Therapeutic Benefit ◽

Transcriptional Elongation ◽

Transcriptional Reprogramming ◽

Basal Expression ◽

Development And Differentiation

The nuclear factor-κB (NFκB) family of transcription factors has been implicated in inflammatory disorders, viral infections, and cancer. Most of the drugs that inhibit NFκB show significant side effects, possibly due to sustained NFκB suppression. Drugs affecting induced, but not basal, NFκB activity may have the potential to provide therapeutic benefit without associated toxicity. NFκB activation by stress-inducible cell cycle inhibitor p21 was shown to be mediated by a p21-stimulated transcription-regulating kinase CDK8. CDK8 and its paralog CDK19, associated with the transcriptional Mediator complex, act as coregulators of several transcription factors implicated in cancer; CDK8/19 inhibitors are entering clinical development. Here we show that CDK8/19 inhibition by different small-molecule kinase inhibitors or shRNAs suppresses the elongation of NFκB-induced transcription when such transcription is activated by p21-independent canonical inducers, such as TNFα. On NFκB activation, CDK8/19 are corecruited with NFκB to the promoters of the responsive genes. Inhibition of CDK8/19 kinase activity suppresses the RNA polymerase II C-terminal domain phosphorylation required for transcriptional elongation, in a gene-specific manner. Genes coregulated by CDK8/19 and NFκB includeIL8,CXCL1, andCXCL2, which encode tumor-promoting proinflammatory cytokines. Although it suppressed newly induced NFκB-driven transcription, CDK8/19 inhibition in most cases had no effect on the basal expression of NFκB-regulated genes or promoters; the same selective regulation of newly induced transcription was observed with other transcription signals potentiated by CDK8/19. This selective role of CDK8/19 identifies these kinases as mediators of transcriptional reprogramming, a key aspect of development and differentiation as well as pathological processes.

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