Inherited blood cancer predisposition through altered transcription elongation.

Despite advances in defining diverse somatic mutations that cause myeloid malignancies, a significant heritable component for these cancers remains largely unexplained. Here, we perform rare variant association studies in a large population cohort to identify inherited predisposition genes for these blood cancers. CTR9, which encodes a key component of the PAF1 transcription elongation complex, is among the significant genes identified. The risk variants found in the cases cause loss of function and result in a ∼10-fold increased odds of acquiring a myeloid malignancy. Partial CTR9 loss of function expands human hematopoietic stem cells (HSCs) by increased super elongation complex-mediated transcriptional activity, which thereby increases the expression of key regulators of HSC self-renewal. By following up on insights from a human genetic study examining inherited predisposition to the myeloid malignancies, we define a previously unknown antagonistic interaction between the PAF1 and super elongation complexes. These insights could enable targeted approaches for blood cancer prevention.

Rtf1-dependent transcriptional pausing regulates cardiogenesis.

During heart development, a well-characterized network of transcription factors initiates cardiac gene expression and defines the precise timing and location of cardiac progenitor specification. However, our understanding of the post-initiation transcriptional events that regulate cardiac gene expression is still incomplete. The PAF1C component Rtf1 is a transcription regulatory protein that modulates pausing and elongation of RNA Pol II, as well as cotranscriptional histone modifications. Here we report that Rtf1 is essential for cardiogenesis in fish and mammals, and that in the absence of Rtf1 activity, cardiac progenitors arrest in an immature state. We found that Rtf1's Plus3 domain, which confers interaction with the transcriptional pausing and elongation regulator Spt5, was necessary for cardiac progenitor formation. ChIP-seq analysis further revealed changes in the occupancy of RNA Pol II around the transcription start site (TSS) of cardiac genes in rtf1 morphants reflecting a reduction in transcriptional pausing. Intriguingly, inhibition of pause release in rtf1 morphants and mutants restored the formation of cardiac cells and improved Pol II occupancy at the TSS of key cardiac genes. Our findings highlight the crucial role that transcriptional pausing plays in promoting normal gene expression levels in a cardiac developmental context.

Aberrant accumulation of Kras-dependent pervasive transcripts during tumor progression renders cancer cells dependent on PAF1 expression.

KRAS is the most commonly mutated oncogene in human cancer, and mutant KRAS is responsible for over 90% of pancreatic ductal adenocarcinoma (PDAC), the most lethal cancer. Here, we show that RNA polymerase II-associated factor 1 complex (PAF1C) is specifically required for survival of PDAC but not normal adult pancreatic cells. We show that PAF1C maintains cancer cell genomic stability by restraining overaccumulation of enhancer RNAs (eRNAs) and promoter upstream transcripts (PROMPTs) driven by mutant Kras. Loss of PAF1C leads to cancer-specific lengthening and accumulation of pervasive transcripts on chromatin and concomitant aberrant R-loop formation and DNA damage, which, in turn, trigger cell death. We go on to demonstrate that the global transcriptional hyperactivation driven by Kras signaling during tumorigenesis underlies the specific demand for PAF1C by cancer cells. Our work provides insights into how enhancer transcription hyperactivation causes general transcription factor addiction during tumorigenesis.

Beyond gene expression: how MYC relieves transcription stress.

MYC oncoproteins are key drivers of tumorigenesis. As transcription factors, MYC proteins regulate transcription by all three nuclear polymerases and gene expression. Accumulating evidence shows that MYC proteins are also crucial for enhancing the stress resilience of transcription. MYC proteins relieve torsional stress caused by active transcription, prevent collisions between the transcription and replication machineries, resolve R-loops, and repair DNA damage by participating in a range of protein complexes and forming multimeric structures at sites of genomic instability. We review the key complexes and multimerization properties of MYC proteins that allow them to mitigate transcription-associated DNA damage, and propose that the oncogenic functions of MYC extend beyond the modulation of gene expression.

Paf1 complex subunit Rtf1 stimulates H2B ubiquitylation by interacting with the highly conserved N-terminal helix of Rad6.

Histone modifications coupled to transcription elongation play important roles in regulating the accuracy and efficiency of gene expression. The monoubiquitylation of a conserved lysine in H2B (K123 in Saccharomyces cerevisiae; K120 in humans) occurs cotranscriptionally and is required for initiating a histone modification cascade on active genes. H2BK123 ubiquitylation (H2BK123ub) requires the RNA polymerase II (RNAPII)-associated Paf1 transcription elongation complex (Paf1C). Through its histone modification domain (HMD), the Rtf1 subunit of Paf1C directly interacts with the ubiquitin conjugase Rad6, leading to the stimulation of H2BK123ub in vivo and in vitro. To understand the molecular mechanisms that target Rad6 to its histone substrate, we identified the site of interaction for the HMD on Rad6. Using in vitro cross-linking followed by mass spectrometry, we localized the primary contact surface for the HMD to the highly conserved N-terminal helix of Rad6. Using a combination of genetic, biochemical, and in vivo protein cross-linking experiments, we characterized separation-of-function mutations in S. cerevisiae RAD6 that greatly impair the Rad6-HMD interaction and H2BK123 ubiquitylation but not other Rad6 functions. By employing RNA-sequencing as a sensitive approach for comparing mutant phenotypes, we show that mutating either side of the proposed Rad6-HMD interface yields strikingly similar transcriptome profiles that extensively overlap with those of a mutant that lacks the site of ubiquitylation in H2B. Our results fit a model in which a specific interface between a transcription elongation factor and a ubiquitin conjugase guides substrate selection toward a highly conserved chromatin target during active gene expression.

Rtf1 Transcriptionally Regulates Neonatal and Adult Cardiomyocyte Biology.

The PAF1 complex component Rtf1 is an RNA Polymerase II-interacting transcription regulatory protein that promotes transcription elongation and the co-transcriptional monoubiquitination of histone 2B. Rtf1 plays an essential role in the specification of cardiac progenitors from the lateral plate mesoderm during early embryogenesis, but its requirement in mature cardiac cells is unknown. Here, we investigate the importance of Rtf1 in neonatal and adult cardiomyocytes using knockdown and knockout approaches. We demonstrate that loss of Rtf1 activity in neonatal cardiomyocytes disrupts cell morphology and results in a breakdown of sarcomeres. Similarly, Rtf1 ablation in mature cardiomyocytes of the adult mouse heart leads to myofibril disorganization, disrupted cell-cell junctions, fibrosis, and systolic dysfunction. Rtf1 knockout hearts eventually fail and exhibit structural and gene expression defects resembling dilated cardiomyopathy. Intriguingly, we observed that loss of Rtf1 activity causes a rapid change in the expression of key cardiac structural and functional genes in both neonatal and adult cardiomyocytes, suggesting that Rtf1 is continuously required to support expression of the cardiac gene program.

Structural Basis of the Transcriptional Elongation Factor Paf1 Core Complex from Saccharomyces eubayanus.

The multicomponent polymerase associated factor 1 (Paf1) complex (PAF1C) is an important transcription elongation factor that upregulates RNA polymerase II-mediated genome-wide transcription. PAF1C can regulate transcription through direct association with the polymerase or by impacting the chromatin structure epigenetically. In recent years, significant progress has been made in understanding the molecular mechanisms of PAF1C. However, high-resolution structures that can clarify the interaction details among the components of the complex are still needed. In this study, we evaluated the structural core of the yeast PAF1C containing the four components Ctr9, Paf1, Cdc73 and Rtf1 at high resolution. We observed the interaction details among these components. In particular, we identified a new binding surface of Rtf1 on PAF1C and found that the C-terminal sequence of Rtf1 dramatically changed during evolution, which may account for its different binding affinities to PAF1C among species. Our work presents a precise model of PAF1C, which will facilitate our understanding of the molecular mechanism and the in vivo function of the yeast PAF1C.

Coordinated histone variant H2A.Z eviction and H3.3 deposition control plant thermomorphogenesis.

Plants can sense temperature changes and adjust their development and morphology accordingly in a process called thermomorphogenesis. This phenotypic plasticity implies complex mechanisms regulating gene expression reprogramming in response to environmental alteration. Histone variants often associate with specific chromatin states; yet, how their deposition/eviction modulates transcriptional changes induced by environmental cues remains elusive. In Arabidopsis thaliana, temperature elevation-induced transcriptional activation at thermo-responsive genes entails the chromatin eviction of a histone variant H2A.Z by INO80, which is recruited to these loci via interacting with a key thermomorphogenesis regulator PIF4. Here, we show that both INO80 and the deposition chaperones of another histone variant H3.3 associate with ELF7, a critical component of the transcription elongator PAF1 complex. H3.3 promotes thermomorphogenesis and the high temperature-enhanced RNA Pol II transcription at PIF4 targets, and it is broadly required for the H2A.Z removal-induced gene activation. Reciprocally, INO80 and ELF7 regulate H3.3 deposition, and are necessary for the high temperature-induced H3.3 enrichment at PIF4 targets. Our findings demonstrate close coordination between H2A.Z eviction and H3.3 deposition in gene activation induced by high temperature, and pinpoint the importance of histone variants dynamics in transcriptional regulation.

Role of the Paf1 complex in the maintenance of stem cell pluripotency and development.

Cell identity is determined by the transcriptional regulation of a cell-type-specific gene group. The Paf1 complex (Paf1C), an RNA polymerase II-associating factor, is an important transcriptional regulator that not only participates in transcription elongation and termination but also affects transcription-coupled histone modifications and chromatin organisation. Recent studies have shown that Paf1C is involved in the expression of genes required for self-renewal and pluripotency in stem cells and tumorigenesis. In this review, we focused on the role of Paf1C as a critical transcriptional regulator in cell fate decisions. Paf1C affects the pluripotency of stem cells by regulating the expression of core transcription factors such as Oct4 and Nanog. In addition, Paf1C directly binds to the promoters or distant elements of target genes, thereby maintaining the pluripotency in embryonic stem cells derived from an early stage of the mammalian embryo. Paf1C is upregulated in cancer stem cells, as compared with that in cancer cells, suggesting that Paf1C may be a target for cancer therapy. Interestingly, Paf1C is involved in multiple developmental stages in Drosophila, zebrafish, mice and even humans, thereby displaying a trend for the correlation between Paf1C and cell fate. Thus, we propose that Paf1C is a critical contributor to cell differentiation, cell specification and its characteristics and could be employed as a therapeutic target in developmental diseases.

The Paf1 complex is required for RNA polymerase II removal in response to DNA damage.

Rpb1, the largest subunit of RNA polymerase II (RNAPII), is rapidly polyubiquitinated and degraded in response to DNA damage; this process is considered to be a "mechanism of last resort'' employed by cells. The underlying mechanism of this process remains elusive. Here, we uncovered a previously uncharacterized multistep pathway in which the polymerase-associated factor 1 (Paf1) complex (PAF1C, composed of the subunits Ctr9, Paf1, Leo1, Cdc73, and Rtf1) is involved in regulating the RNAPII pool by stimulating Elongin-Cullin E3 ligase complex-mediated Rpb1 polyubiquitination and subsequent degradation by the proteasome following DNA damage. Mechanistically, Spt5 is dephosphorylated following DNA damage, thereby weakening the interaction between the Rtf1 subunit and Spt5, which might be a key step in initiating Rpb1 degradation. Next, Rad26 is loaded onto stalled RNAPII to replace the Spt4/Spt5 complex in an RNAPII-dependent manner and, in turn, recruits more PAF1C to DNA lesions via the binding of Rad26 to the Leo1 subunit. Importantly, the PAF1C, assembled in a Ctr9-mediated manner, coordinates with Rad26 to localize the Elongin-Cullin complex on stalled RNAPII, thereby inducing RNAPII removal, in which the heterodimer Paf1/Leo1 and the subunit Cdc73 play important roles. Together, our results clearly revealed a new role of the intact PAF1C in regulating the RNAPII pool in response to DNA damage.

SHP-1 dephosphorylates histone H2B to facilitate its ubiquitination during transcription.

Dynamic regulation of phosphorylation and dephosphorylation of histones is essential for eukaryotic transcription, but the enzymes engaged in histone dephosphorylation are not fully explored. Here, we show that the tyrosine phosphatase SHP-1 dephosphorylates histone H2B and plays a critical role during transition from the initiation to the elongation stage of transcription. Nuclear-localized SHP-1 is associated with the Paf1 complex at chromatin and dephosphorylates H2B at tyrosine 121. Moreover, knockout of SHP-1, or expression of a mutant mimicking constitutive phosphorylation of H2B Y121, leads to a reduction in genome-wide H2B ubiquitination, which subsequently causes defects in RNA polymerase II-dependent transcription. Mechanistically, we demonstrate that Y121 phosphorylation precludes H2B's interaction with the E2 enzyme, indicating that SHP-1-mediated dephosphorylation of this residue may be a prerequisite for efficient H2B ubiquitination. Functionally, we find that SHP-1-mediated H2B dephosphorylation contributes to maintaining basal autophagic flux in cells through the efficient transcription of autophagy and lysosomal genes. Collectively, our study reveals an important modification of histone H2B regulated by SHP-1 that has a role during eukaryotic transcription.

Cap-binding complex assists RNA polymerase II transcription in plant salt stress response.

Adaptive response to stress involves an extensive reprogramming of gene expression. Under stressful conditions, the induction of efficient changes in messenger RNA (mRNA) production is crucial for maximized plant survival. Transcription and pre-mRNA processing are two closely related steps in mRNA biogenesis, yet how they are controlled in plant stress response remains elusive. Here, we show that the Arabidopsis nuclear cap-binding complex (CBC) component CBP20 directly interacts with ELF7, a subunit of the transcription elongation factor RNA Pol II-associated factor 1 complex (PAF1c) to promote RNA Pol II transcription in plant response to salt stress. CBP20 and ELF7 coregulate the expression of a large number of genes including those crucial for salt tolerance. Both CBP20 and ELF7 are required for enhanced RNA Pol II elongation at salt-activated genes. Though CBP20 also regulates intron splicing, this function is largely independent of ELF7. Our study reveals the function of an RNA processing regulator CBC in assisting efficient RNA Pol II transcription and pinpoints the complex roles of CBC on mRNA production in plant salt stress resistance.

The PAF1 complex promotes 3' processing of pervasive transcripts.

The PAF1 complex (PAF1C) functions in multiple transcriptional processes involving RNA polymerase II (RNA Pol II). Enhancer RNAs (eRNAs) and promoter upstream transcripts (PROMPTs) are pervasive transcripts transcribed by RNA Pol II and degraded rapidly by the nuclear exosome complex after 3' endonucleolytic cleavage by the Integrator complex (Integrator). Here we show that PAF1C has a role in termination of eRNAs and PROMPTs that are cleaved 1-3 kb downstream of the transcription start site. Mechanistically, PAF1C facilitates recruitment of Integrator to sites of pervasive transcript cleavage, promoting timely cleavage and transcription termination. We also show that PAF1C recruits Integrator to coding genes, where PAF1C then dissociates from Integrator upon entry into processive elongation. Our results demonstrate a function of PAF1C in limiting the length and accumulation of pervasive transcripts that result from non-productive transcription.

Crystal Structure of the Core Module of the Yeast Paf1 Complex.

The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), which consists of five core subunits: Ctr9, Paf1, Leo1, Cdc73, and Rtf1, acts as a diverse hub that regulates all stages of RNA polymerase II-mediated transcription and various other cellular functions. However, the underlying mechanisms remain unclear. Here, we report the crystal structure of the core module derived from a quaternary Ctr9/Paf1/Cdc73/Rtf1 complex of S. cerevisiae PAF1C, which reveals interfaces between the tetratricopeptide repeat module in Ctr9 and Cdc73 or Rtf1, and find that the Ctr9/Paf1 subcomplex is the key scaffold for PAF1C assembly. Our study demonstrates that Cdc73 binds Ctr9/Paf1 subcomplex with a very similar conformation within thermophilic fungi or human PAF1C, and that the binding of Cdc73 to PAF1C is important for yeast growth. Importantly, our structure reveals for the first time that the extreme C-terminus of Rtf1 adopts an "L"-shaped structure, which interacts with Ctr9 specifically. In addition, disruption of the binding of either Cdc73 or Rtf1 to PAF1C greatly affects the normal level of histone H2B K123 monoubiquitination in vivo. Collectively, our results provide a structural insight into the architecture of the quaternary Ctr9/Paf1/Cdc73/Rtf1 complex and PAF1C functional regulation.

Opposing functions of the Hda1 complex and histone H2B mono-ubiquitylation in regulating cryptic transcription in Saccharomyces cerevisiae.

Maintenance of chromatin structure under the disruptive force of transcription requires cooperation among numerous regulatory factors. Histone post-translational modifications can regulate nucleosome stability and influence the disassembly and reassembly of nucleosomes during transcription elongation. The Paf1 transcription elongation complex, Paf1C, is required for several transcription-coupled histone modifications, including the mono-ubiquitylation of H2B. In Saccharomyces cerevisiae, amino acid substitutions in the Rtf1 subunit of Paf1C greatly diminish H2B ubiquitylation and cause transcription to initiate at a cryptic promoter within the coding region of the FLO8 gene, an indicator of chromatin disruption. In a genetic screen to identify factors that functionally interact with Paf1C, we identified mutations in HDA3, a gene encoding a subunit of the Hda1C histone deacetylase (HDAC), as suppressors of an rtf1 mutation. Absence of Hda1C also suppresses the cryptic initiation phenotype of other mutants defective in H2B ubiquitylation. The genetic interactions between Hda1C and the H2B ubiquitylation pathway appear specific: loss of Hda1C does not suppress the cryptic initiation phenotypes of other chromatin mutants and absence of other HDACs does not suppress the absence of H2B ubiquitylation. Providing further support for an appropriate balance of histone acetylation in regulating cryptic initiation, absence of the Sas3 histone acetyltransferase elevates cryptic initiation in rtf1 mutants. Our data suggest that the H2B ubiquitylation pathway and Hda1C coordinately regulate chromatin structure during transcription elongation and point to a potential role for a HDAC in supporting chromatin accessibility.

Biochemical insights into Paf1 complex-induced stimulation of Rad6/Bre1-mediated H2B monoubiquitination.

The highly conserved multifunctional polymerase-associated factor 1 (Paf1) complex (PAF1C), composed of five core subunits Paf1, Leo1, Ctr9, Cdc73, and Rtf1, participates in all stages of transcription and is required for the Rad6/Bre1-mediated monoubiquitination of histone H2B (H2Bub). However, the molecular mechanisms underlying the contributions of the PAF1C subunits to H2Bub are not fully understood. Here, we report that Ctr9, acting as a hub, interacts with the carboxyl-terminal acidic tail of Rad6, which is required for PAF1C-induced stimulation of H2Bub. Importantly, we found that the Ras-like domain of Cdc73 has the potential to accelerate ubiquitin discharge from Rad6 and thus facilitates H2Bub, a process that might be conserved from yeast to humans. Moreover, we found that Rtf1 HMD stimulates H2Bub, probably through accelerating ubiquitin discharge from Rad6 alone or in cooperation with Cdc73 and Bre1, and that the Paf1/Leo1 heterodimer in PAF1C specifically recognizes the histone H3 tail of nucleosomal substrates, stimulating H2Bub. Collectively, our biochemical results indicate that intact PAF1C is required to efficiently stimulate Rad6/Bre1-mediated H2Bub.

A BRD4-mediated elongation control point primes transcribing RNA polymerase II for 3'-processing and termination.

Transcription elongation has emerged as a regulatory hub in gene expression of metazoans. A major control point occurs during early elongation before RNA polymerase II (Pol II) is released into productive elongation. Prior research has linked BRD4 with transcription elongation. Here, we use rapid BET protein and BRD4-selective degradation along with quantitative genome-wide approaches to investigate direct functions of BRD4 in Pol II transcription regulation. Notably, as an immediate consequence of acute BRD4 loss, promoter-proximal pause release is impaired, and transcriptionally engaged Pol II past this checkpoint undergoes readthrough transcription. An integrated proteome-wide analysis uncovers elongation and 3'-RNA processing factors as core BRD4 interactors. BRD4 ablation disrupts the recruitment of general 3'-RNA processing factors at the 5'-control region, which correlates with RNA cleavage and termination defects. These studies, performed in human cells, reveal a BRD4-mediated checkpoint and begin to establish a molecular link between 5'-elongation control and 3'-RNA processing.

Old factors, new players: transcriptional regulation of autophagy.

Macroautophagy/autophagy is a catabolic process that allows cells to adapt to environmental changes and maintain energy homeostasis. This multistep process is regulated at several levels, including transcriptionally regulating autophagy-related (ATG) gene expression through the action of transcription regulators. Very recently, Wen et al. and we have provided more evidence that two well-known transcription factors regulate different ATG genes to control either nonselective or selective forms of autophagy, respectively. Under nitrogen-starvation conditions, the Spt4-Spt5 complex derepresses ATG8 and ATG41 expression and upregulates bulk autophagy activity. By contrast, under glucose-starvation conditions, the Paf1 complex (the polymerase-associated factor 1 complex, Paf1C) specifically modulates expression of ATG11 and ATG32 to regulate mitophagy. These studies suggest the potential existence of other transcription regulators yet to be discovered that function in the regulation of diverse autophagy pathways.Abbreviations: AMPK: AMP-activated protein kinase; ATG: autophagy-related; NELF: negative elongation factor; Paf1C/PAF1C: polymerase-associated factor 1 complex; RNAP II: RNA polymerase II; Rpd3L: Rpd3 large complex.

The Paf1 complex transcriptionally regulates the mitochondrial-anchored protein Atg32 leading to activation of mitophagy.

Mitophagy is a critical process that safeguards mitochondrial quality control in order to maintain proper cellular homeostasis. Although the mitochondrial-anchored receptor Atg32-mediated cargo-recognition system has been well characterized to be essential for this process, the signaling pathway modulating its expression as a contribution of governing the mitophagy process remains largely unknown. Here, bioinformatics analyses of epigenetic or transcriptional regulators modulating gene expression allow us to identify the Paf1 complex (the polymerase-associated factor 1 complex, Paf1C) as a transcriptional repressor of ATG genes. We show that Paf1C suppresses glucose starvation-induced autophagy, but does not affect nitrogen starvation- or rapamycin-induced autophagy. Moreover, we show that Paf1C specifically regulates mitophagy through modulating ATG32 expression. Deletion of the genes encoding two core subunits of Paf1C, Paf1 and Ctr9, increases ATG32 and ATG11 expression and facilitates mitophagy activity. Although Paf1C is required for many histone modifications and gene activation, we show that Paf1C regulates mitophagy independent of its positive regulatory role in other processes. More importantly, we also demonstrate the mitophagic role of PAF1C in mammals. Overall, we conclude that Paf1C maintains mitophagy at a low level through binding the promoter of the ATG32 gene in glucose-rich conditions. Dissociation of Paf1C from ATG32 leads to the increased expression of this gene, and mitophagy induction upon glucose starvation. Thus, we uncover a new role of Paf1C in modulating the mitophagy process at the transcriptional level. ABBREVIATIONS: AMPK: AMP-activated protein kinase; ATP5F1A: ATP synthase F1 subunit alpha; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CCCP: chlorophenylhydrazone; DFP: chelator deferiprone; GFP: green fluorescent protein; H2B-Ub1: H2B monoubiquitination; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; KD: kinase dead; OPTN, optineurin; Paf1: polymerase-associated factor 1; PINK1: PTEN induced kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; RT-qPCR: real-time quantitative PCR; SD-N: synthetic dropout without nitrogen base; TIMM23: translocase of inner mitochondrial membrane 23; TOMM20: translocase of outer mitochondrial membrane 20; WT: wild-type; YPD: yeast extract peptone dextrose; YPL: yeast extract peptone lactate.