CTR9, a Component of PAF Complex, Controls Elongation Block at the c-Fos Locus via Signal-Dependent Regulation of Chromatin-Bound NELF Dissociation

The PAF complex (PAFC) coordinates transcription elongation and mRNA processing and its CDC73/parafibromin subunit functions as a tumour suppressor. The NF2/Merlin tumour suppressor functions both at the cell cortex and nucleus and is a key mediator of contact inhibition but the molecular mechanisms remain unclear. In this study we have used affinity proteomics to identify novel Merlin interacting proteins and show that Merlin forms a complex with multiple proteins involved in RNA processing including the PAFC and the CHD1 chromatin remodeller. Tumour-derived inactivating mutations in both Merlin and the CDC73 PAFC subunit mutually disrupt their interaction and growth suppression by Merlin requires CDC73. Merlin interacts with the PAFC in a cell density-dependent manner and we identify a role for FAT cadherins in regulating the Merlin-PAFC interaction. Our results suggest that in addition to its function within the Hippo pathway, Merlin is part of a tumour suppressor network regulated by cell-cell adhesion which coordinates post-initiation steps of the transcription cycle of genes mediating contact inhibition.

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Control of transcriptional elongation and cotranscriptional histone modification by the yeast BUR kinase substrate Spt5

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0806302106 ◽

2009 ◽

Vol 106 (17) ◽

pp. 6956-6961 ◽

Cited By ~ 95

Author(s):

Karen Zhou ◽

Wei Hung William Kuo ◽

Jeffrey Fillingham ◽

Jack F. Greenblatt

Keyword(s):

Histone Modification ◽

Rna Polymerase Ii ◽

Transcription Elongation ◽

Consensus Sequence ◽

Elongation Factor ◽

Transcriptional Elongation ◽

Histone H2b ◽

Paf Complex

Elongation by RNA polymerase II (RNAPII) is a finely regulated process in which many elongation factors contribute to gene regulation. Among these factors are the polymerase-associated factor (PAF) complex, which associates with RNAPII, and several cyclin-dependent kinases, including positive transcription elongation factor b (P-TEFb) in humans and BUR kinase (Bur1–Bur2) and C-terminal domain (CTD) kinase 1 (CTDK1) in Saccharomyces cerevisiae. An important target of P-TEFb and CTDK1, but not BUR kinase, is the CTD of the Rpb1 subunit of RNAPII. Although the essential BUR kinase phosphorylates Rad6, which is required for histone H2B ubiquitination on K123, Rad6 is not essential, leaving a critical substrate(s) of BUR kinase unidentified. Here we show that BUR kinase is important for the phosphorylation in vivo of Spt5, a subunit of the essential yeast RNAPII elongation factor Spt4/Spt5, whose human orthologue is DRB sensitivity-inducing factor. BUR kinase can also phosphorylate the C-terminal region (CTR) of Spt5 in vitro. Like BUR kinase, the Spt5 CTR is important for promoting elongation by RNAPII and recruiting the PAF complex to transcribed regions. Also like BUR kinase and the PAF complex, the Spt5 CTR is important for histone H2B K123 monoubiquitination and histone H3 K4 and K36 trimethylation during transcription elongation. Our results suggest that the Spt5 CTR, which contains 15 repeats of a hexapeptide whose consensus sequence is S[T/A]WGG[A/Q], is a substrate of BUR kinase and a platform for the association of proteins that promote both transcription elongation and histone modification in transcribed regions.

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Parafibromin, a Component of the Human PAF Complex, Regulates Growth Factors and Is Required for Embryonic Development and Survival in Adult Mice

Molecular and Cellular Biology ◽

10.1128/mcb.00654-07 ◽

2008 ◽

Vol 28 (9) ◽

pp. 2930-2940 ◽

Cited By ~ 65

Author(s):

Pengfei Wang ◽

Michael R. Bowl ◽

Stephanie Bender ◽

Jun Peng ◽

Leslie Farber ◽

...

Keyword(s):

Growth Factors ◽

Gene Mutations ◽

Mammalian Development ◽

Embryonic Fibroblasts ◽

Growth And Survival ◽

Reverse Transcription Pcr ◽

Adult Mice ◽

Regulation Of Growth ◽

Paf Complex

ABSTRACT Parafibromin, a transcription factor associated with the PAF complex, is encoded by the HRPT2 gene, mutations of which cause the hyperparathyroidism-jaw tumor syndrome (OMIM145001). To elucidate the function of parafibromin, we generated conventional and conditional Hrpt2 knockout mice and found that Hrpt2 −/− mice were embryonic lethal by embryonic day 6.5 (E6.5). Controlled deletion of Hrpt2 after E8.5 resulted in apoptosis and growth retardation. Deletion of Hrpt2 in adult mice led to severe cachexia and death within 20 days. To explore the mechanism underlying the embryonic lethality and death of adult mice, mouse embryonic fibroblasts (MEFs) were cultured and Hrpt2 was deleted in vitro. Hrpt2 −/− MEFs underwent apoptosis, while Hrpt2 +/+ and Hrpt2 +/− MEFs grew normally. To study the mechanism of this apoptosis, Hrpt2 +/+ and Hrpt2 −/− MEFs were used in cDNA microarray, semiquantitative reverse transcription-PCR, and chromatin immunoprecipitation assays to identify genes regulated by parafibromin. These revealed that Hrpt2 expression and the parafibromin/PAF complex directly regulate genes involved in cell growth and survival, including H19, Igf1, Igf2, Igfbp4, Hmga1, Hmga2, and Hmgcs2. Thus, our results show that expression of Hrpt2 and parafibromin is pivotal in mammalian development and survival in adults and that these functions are likely mediated by the transcriptional regulation of growth factors.

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Global regulation of heterochromatin spreading by Leo1

Open Biology ◽

10.1098/rsob.150045 ◽

2015 ◽

Vol 5 (5) ◽

pp. 150045 ◽

Cited By ~ 27

Author(s):

Laure Verrier ◽

Francesca Taglini ◽

Ramon R. Barrales ◽

Shaun Webb ◽

Takeshi Urano ◽

...

Keyword(s):

Chromatin Modification ◽

Specific Role ◽

Eukaryotic Genome ◽

Global Regulation ◽

Genome Regulation ◽

H4k16 Acetylation ◽

Paf Complex

Heterochromatin plays important roles in eukaryotic genome regulation. However, the repressive nature of heterochromatin combined with its propensity to self-propagate necessitates robust mechanisms to contain heterochromatin within defined boundaries and thus prevent silencing of expressed genes. Here we show that loss of the PAF complex (PAFc) component Leo1 compromises chromatin boundaries, resulting in invasion of heterochromatin into flanking euchromatin domains. Similar effects are seen upon deletion of other PAFc components, but not other factors with related functions in transcription-associated chromatin modification, indicating a specific role for PAFc in heterochromatin regulation. Loss of Leo1 results in reduced levels of H4K16 acetylation at boundary regions, while tethering of the H4K16 acetyltransferase Mst1 to boundary chromatin suppresses heterochromatin spreading in leo1Δ cells, suggesting that Leo1 antagonises heterochromatin spreading by promoting H4K16 acetylation. Our findings reveal a previously undescribed role for PAFc in regulating global heterochromatin distribution.

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A role for the RNA pol II–associated PAF complex in AID-induced immune diversification

Journal of Experimental Medicine ◽

10.1084/jem.20112145 ◽

2012 ◽

Vol 209 (11) ◽

pp. 2099-2111 ◽

Cited By ~ 47

Author(s):

Katharina L. Willmann ◽

Sara Milosevic ◽

Siim Pauklin ◽

Kerstin-Maike Schmitz ◽

Gopinath Rangam ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Protein Complexes ◽

Chromatin Modification ◽

Cytosine Deamination ◽

Rna Pol Ii ◽

Pol Ii ◽

Antibody Diversification ◽

Dna Instability ◽

Paf Complex

Antibody diversification requires the DNA deaminase AID to induce DNA instability at immunoglobulin (Ig) loci upon B cell stimulation. For efficient cytosine deamination, AID requires single-stranded DNA and needs to gain access to Ig loci, with RNA pol II transcription possibly providing both aspects. To understand these mechanisms, we isolated and characterized endogenous AID-containing protein complexes from the chromatin of diversifying B cells. The majority of proteins associated with AID belonged to RNA polymerase II elongation and chromatin modification complexes. Besides the two core polymerase subunits, members of the PAF complex, SUPT5H, SUPT6H, and FACT complex associated with AID. We show that AID associates with RNA polymerase-associated factor 1 (PAF1) through its N-terminal domain, that depletion of PAF complex members inhibits AID-induced immune diversification, and that the PAF complex can serve as a binding platform for AID on chromatin. A model is emerging of how RNA polymerase II elongation and pausing induce and resolve AID lesions.

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