MYC dephosphorylation by the PP1/PNUTS phosphatase complex regulates chromatin binding and protein stability

Dingar, Dharmendra; Tu, William B.; Resetca, Diana; Lourenco, Corey; Tamachi, Aaliya; Melo, Jason De; Houlahan, Kathleen E.; Kalkat, Manpreet; Chan, Pak-Kei; Boutros, Paul C.; Raught, Brian; Penn, Linda Z.

doi:10.1038/s41467-018-05660-0

Cited by 48 publications

(64 citation statements)

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“…Similar to the mammalian complex, we propose PNUTS is a scaffolding protein for the entire PJW/PP1 complex and regulates PP1 function via the PP1 binding RVxF motif. Only three substrates have been identified for PNUTS/PP1: the Pol II elongation factor Spt5, the CTD of Pol II and MYC [2,60,97,98]. MYC dephosphorylation by PP1 regulates chromatin binding and stability.…”

Section: Discussionmentioning

confidence: 99%

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

et al. 2020

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Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of transcription start sites, suggesting an additional role of the complex in regulating termination of bi-directional transcription. In addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes involved in host immune evasion. Our results suggest a novel mechanistic link between base J and Pol II polycistronic transcription termination in kinetoplastids. Author summaryTrypanosoma brucei is a parasitic protozoan that causes African sleeping sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain multiple genes that are co-transcribed from a single promoter. We have recently described the presence of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene clusters where the loss of base J and H3.V leads to read-through transcription and the expression of downstream genes. We now PLOS Genetics | https://doi.identify a novel stable multimeric complex containing a J binding protein (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex regulates termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of specific proteins (including Pol II and termination factors) by PP1. These findings significantly expand our understanding of mechanisms underlying transcription termination in eukaryotes, including organisms that utilize polycistronic transcription and novel epigenetic marks...

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Section: Discussionmentioning

confidence: 99%

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

et al. 2020

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“…The MYC-MIZ-1 interaction accounts for 25%-40% of MYC-repressed genes (Walz et al, 2014), suggesting additional interactors contribute to transcriptional repression. To characterize the MYC interactome, we recently used an in-cell biotin-labeling mass spectrometry technique called MYC-BioID that captured known MYC-interacting chromatin complexes, including the TRRAP-HAT complex, and additional complexes enriched for epigenetic functions (Dingar et al, 2015(Dingar et al, , 2018. Epigenetic proteins have emerged as critical regulators of MYC at multiple levels to control its transcriptional activity (Poole and van Riggelen, 2017).…”

Section: Significancementioning

confidence: 99%

“…Among the MYC interactors, we identified from MYC-BioID in HeLa cells, we prioritized those involved in cancer and targetable with small-molecule inhibitors (Dingar et al, 2018). This revealed the G9a methyltransferase and other proteins of the G9a complex, including CDYL (Mulligan et al, 2008) and WIZ (Ueda et al, 2006), as high confidence MYC interactors (significance analysis of interactome score > 0.80) ( Figure 1A).…”

Section: Myc Interacts With G9a and Regulates H3k9me2 Levelsmentioning

confidence: 99%

“…MYC induction alone (A) BioID of BirA control and BirA-MYC performed in HeLa. Total peptide counts of select proteins from two biological replicates with two technical replicates are extracted from the full dataset in Dingar et al (2018). Significant interactors have significance analysis of interactome (SAINT) score > 0.80.…”

Section: G9a Binds To and Regulates Myc-repressed Target Genesmentioning

confidence: 99%

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MYC Interacts with the G9a Histone Methyltransferase to Drive Transcriptional Repression and Tumorigenesis

Shiah

Lourenco

et al. 2018

Cancer Cell

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Graphical Abstract Highlights d MYC interacts with and regulates the G9a histone methyltransferase complex d G9a is required for MYC chromatin binding and gene repression d MYC-G9a interaction requires the transformation-dependent MYC box II region d G9a is a synthetic lethal vulnerability in MYC-dependent basal breast cancer SUMMARYMYC is an oncogenic driver that regulates transcriptional activation and repression. Surprisingly, mechanisms by which MYC promotes malignant transformation remain unclear. We demonstrate that MYC interacts with the G9a H3K9-methyltransferase complex to control transcriptional repression. Inhibiting G9a hinders MYC chromatin binding at MYC-repressed genes and de-represses gene expression. By identifying the MYC box II region as essential for MYC-G9a interaction, a long-standing missing link between MYC transformation and gene repression is unveiled. Across breast cancer cell lines, the anti-proliferative response to G9a pharmacological inhibition correlates with MYC sensitivity and gene signatures. Consistently, genetically depleting G9a in vivo suppresses MYC-dependent tumor growth. These findings unveil G9a as an epigenetic regulator of MYC transcriptional repression and a therapeutic vulnerability in MYC-driven cancers.

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“…Similar to the mammalian complex, we propose PNUTS is a scaffolding protein for the entire PJW/PP1 complex and regulates PP1 function via the PP1 binding RVxF motif. Only three substrates have been identified for PNUTS/PP1: the Pol II elongation factor Spt5, the CTD of Pol II and MYC (2, 53, 91, 92). MYC dephosphorylation by PP1 regulates chromatin binding and stability.…”

Section: Discussionmentioning

confidence: 99%

Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

Sabatini

Kieft

Zhang

et al. 2019

Preprint

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19 Base J, β-D-glucosyl-hydroxymethyluracil, is a modification of thymine DNA base involved in RNA 20 Polymerase (Pol) II transcription termination in kinetoplastid protozoa. Little is understood regarding how 21 specific thymine residues are targeted for J-modification or the mechanism of J regulated transcription 22 termination. To identify proteins involved in J-synthesis, we expressed a tagged version of the J-23 glucosyltransferase (JGT) in Leishmania tarentolae, and identified four co-purified proteins by mass 24 spectrometry: protein phosphatase (PP1), a homolog of Wdr82, a potential PP1 regulatory protein 25 (PNUTS) and a protein containing a J-DNA binding domain (named JBP3). Gel shift studies indicate 26 JBP3 is a J-DNA binding protein. Reciprocal tagging, co-IP and sucrose gradient analyses indicate PP1, 27 JGT, JBP3, Wdr82 and PNUTS form a multimeric complex in kinetoplastids, similar to the mammalian 28 PTW/PP1 complex involved in transcription termination via PP1 mediated dephosphorylation of Pol II. 29 Using RNAi and analysis of Pol II termination by RNA-seq and RT-PCR, we demonstrate that ablation of 30 PNUTS, JBP3 and Wdr82 lead to defects in Pol II termination at the 3'-end of polycistronic gene arrays in 31 Trypanosoma brucei. Mutants also contain increased antisense RNA levels upstream of promoters, 32 suggesting an additional role of the complex in regulating termination of bi-directional transcription. In 33 addition, PNUTS loss causes derepression of silent Variant Surface Glycoprotein genes important for 34 host immune evasion. Our results provide the first direct mechanistic link between base J and regulation 35 of Pol II termination and suggest a novel molecular model for the role of the CTD of Pol II in terminating 36 polycistronic transcription in trypanosomatids. 37 38 Author Summary 39 Trypanosoma brucei is an early-diverged parasitic protozoan that causes African sleeping 40 sickness in humans. The genome of T. brucei is organized into polycistronic gene clusters that contain 41 multiple genes that are co-transcribed from a single promoter. We have recently described the presence 42 of a modified DNA base J and variant of histone H3 (H3.V) at transcription termination sites within gene 43 clusters where the loss of base J and H3.V leads to read-through transcription and the expression of 44 downstream genes. We now identify a novel stable multimeric complex containing a J binding protein . CC-BY 4.0 International license is made available under aThe copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/753004 doi: bioRxiv preprint 45 (JBP3), base J glucosyltransferase (JGT), PP1 phosphatase, PP1 interactive-regulatory protein (PNUTS) 46 and Wdr82, which we refer to as PJW/PP1. A similar complex (PTW/PP1) has been shown to be involved 47 in Pol II termination in humans and yeast. We demonstrate that PNUTS, JBP3 and Wdr82 mutants lead 48 to read-through transcription in T. brucei. Our data suggest the PJW/PP1 complex reg...

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MYC dephosphorylation by the PP1/PNUTS phosphatase complex regulates chromatin binding and protein stability

Cited by 48 publications

References 55 publications

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

Identification of a novel base J binding protein complex involved in RNA polymerase II transcription termination in trypanosomes

MYC Interacts with the G9a Histone Methyltransferase to Drive Transcriptional Repression and Tumorigenesis

Identification of a Novel Base J Binding Protein Complex Involved in RNA Polymerase II Transcription Termination in Trypanosomes

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