The Rtf1/Prf1-dependent histone modification axis counteracts multi-drug resistance in fission yeast.

RNA polymerase II transcription elongation directs an intricate pattern of histone modifications. This pattern includes a regulatory cascade initiated by the elongation factor Rtf1, leading to monoubiquitylation of histone H2B, and subsequent methylation of histone H3 on lysine 4. Previous studies have defined the molecular basis for these regulatory relationships, but it remains unclear how they regulate gene expression. To address this question, we investigated a drug resistance phenotype that characterizes defects in this axis in the model eukaryote Schizosaccharomyces pombe (fission yeast). The mutations caused resistance to the ribonucleotide reductase inhibitor hydroxyurea (HU) that correlated with a reduced effect of HU on dNTP pools, reduced requirement for the S-phase checkpoint, and blunting of the transcriptional response to HU treatment. Mutations in the C-terminal repeat domain of the RNA polymerase II large subunit Rpb1 led to similar phenotypes. Moreover, all the HU-resistant mutants also exhibited resistance to several azole-class antifungal agents. Our results suggest a novel, shared gene regulatory function of the Rtf1-H2Bub1-H3K4me axis and the Rpb1 C-terminal repeat domain in controlling fungal drug tolerance.

Spt5 C-terminal repeat domain phosphorylation and length negatively regulate heterochromatin through distinct mechanisms.

Heterochromatin is a condensed chromatin structure that represses transcription of repetitive DNA elements and developmental genes, and is required for genome stability. Paradoxically, transcription of heterochromatic sequences is required for establishment of heterochromatin in diverse eukaryotic species. As such, components of the transcriptional machinery can play important roles in establishing heterochromatin. How these factors coordinate with heterochromatin proteins at nascent heterochromatic transcripts remains poorly understood. In the model eukaryote Schizosaccharomyces pombe (S. pombe), heterochromatin nucleation can be coupled to processing of nascent transcripts by the RNA interference (RNAi) pathway, or to other post-transcriptional mechanisms that are RNAi-independent. Here we show that the RNA polymerase II processivity factor Spt5 negatively regulates heterochromatin in S. pombe through its C-terminal domain (CTD). The Spt5 CTD is analogous to the CTD of the RNA polymerase II large subunit, and is comprised of multiple repeats of an amino acid motif that is phosphorylated by Cdk9. We provide evidence that genetic ablation of Spt5 CTD phosphorylation results in aberrant RNAi-dependent nucleation of heterochromatin at an ectopic location, as well as inappropriate spread of heterochromatin proximal to centromeres. In contrast, truncation of Spt5 CTD repeat number enhanced RNAi-independent heterochromatin formation and bypassed the requirement for RNAi. We relate these phenotypes to the known Spt5 CTD-binding factor Prf1/Rtf1. This separation of function argues that Spt5 CTD phosphorylation and CTD length restrict heterochromatin through unique mechanisms. More broadly, our findings argue that length and phosphorylation of the Spt5 CTD repeat array have distinct regulatory effects on transcription.

Comparing the signaling and transcriptome profiling landscapes of human iPSC-derived and primary rat neonatal cardiomyocytes.

The inaccessibility of human cardiomyocytes significantly hindered years of cardiovascular research efforts. To overcome these limitations, non-human cell sources were used as proxies to study heart function and associated diseases. Rodent models became increasingly acceptable surrogates to model the human heart either in vivo or through in vitro cultures. More recently, due to concerns regarding animal to human translation, including cross-species differences, the use of human iPSC-derived cardiomyocytes presented a renewed opportunity. Here, we conducted a comparative study, assessing cellular signaling through cardiac G protein-coupled receptors (GPCRs) in rat neonatal cardiomyocytes (RNCMs) and human induced pluripotent stem cell-derived cardiomyocytes. Genetically encoded biosensors were used to explore GPCR-mediated nuclear protein kinase A (PKA) and extracellular signal-regulated kinase 1/ 2 (ERK1/2) activities in both cardiomyocyte populations. To increase data granularity, a single-cell analytical approach was conducted. Using automated high content microscopy, our analyses of nuclear PKA and ERK1/2 signaling revealed distinct response clusters in rat and human cardiomyocytes. In line with this, bulk RNA-seq revealed key differences in the expression patterns of GPCRs, G proteins and downstream effector expression levels. Our study demonstrates that human stem cell-derived models of the cardiomyocyte offer distinct advantages for understanding cellular signaling in the heart.

Gßγ subunits colocalize with RNA polymerase II and regulate transcription in cardiac fibroblasts.

Gßγ subunits mediate many different signaling processes in various compartments of the cell, including the nucleus. To gain insight into the functions of nuclear Gßγ signaling, we investigated the functional role of Gßγ signaling in the regulation of GPCR-mediated gene expression in primary rat neonatal cardiac fibroblasts. We identified a novel, negative, regulatory role for the Gß1γ dimer in the fibrotic response. Depletion of Gß1 led to derepression of the fibrotic response at the mRNA and protein levels under basal conditions and an enhanced fibrotic response after sustained stimulation of the angiotensin II type I receptor. Our genome-wide chromatin immunoprecipitation experiments revealed that Gß1 colocalized and interacted with RNA polymerase II on fibrotic genes in an angiotensin II-dependent manner. Additionally, blocking transcription with inhibitors of Cdk9 prevented association of Gßγ with transcription complexes. Together, our findings suggest that Gß1γ is a novel transcriptional regulator of the fibrotic response that may act to restrict fibrosis to conditions of sustained fibrotic signaling. Our work expands the role for Gßγ signaling in cardiac fibrosis and may have broad implications for the role of nuclear Gßγ signaling in other cell types.

Decoding histone ubiquitylation.

Histone ubiquitylation is a critical part of both active and repressed transcriptional states, and lies at the heart of DNA damage repair signaling. The histone residues targeted for ubiquitylation are often highly conserved through evolution, and extensive functional studies of the enzymes that catalyze the ubiquitylation and de-ubiquitylation of histones have revealed key roles linked to cell growth and division, development, and disease in model systems ranging from yeast to human cells. Nonetheless, the downstream consequences of these modifications have only recently begun to be appreciated on a molecular level. Here we review the structure and function of proteins that act as effectors or "readers" of histone ubiquitylation. We highlight lessons learned about how ubiquitin recognition lends specificity and function to intermolecular interactions in the context of transcription and DNA repair, as well as what this might mean for how we think about histone modifications more broadly.

A role for BET proteins in regulating basal, dopamine-induced and cAMP/PKA-dependent transcription in rat striatal neurons.

The activity of striatal medium-spiny projection neurons is regulated by D1 and D2 dopamine receptors. The D1 receptor (D1R) is a Gαs/olf-coupled GPCR which activates a cAMP/PKA/DARPP-32 signalling cascade that increases excitability and facilitates plasticity, partly through the regulation of transcription. Upon activation via D1R, PKA can translocate to the nucleus to regulate transcription through the phosphorylation of various targets. One candidate effector of PKA-dependent transcriptional regulation is the BET protein Brd4. It is known that when Brd4 is activated by phosphorylation, it binds more readily to acetylated histones at promoters and enhancers; moreover, in non-neuronal cells, PKA signalling has been shown to increase recruitment of Brd4 to chromatin. However, it is unknown whether BET proteins, or Brd4 specifically, are involved in transcriptional activation by cAMP/PKA in neurons. Here, we demonstrate that in adult rats, inhibition of BET proteins with the bromodomain inhibitor JQ1 suppressed the expression of ~25% of D1R-upregulated genes, while also increasing the expression of a subset of immediate-early genes. We further found that cAMP/PKA signalling promotes Brd4 recruitment to dopamine-induced genes in striatal neurons, and that knockdown of Brd4 attenuates D1R-induced gene expression. Finally, we report that JQ1 treatment downregulated expression of many GPCRs and also impaired ERK1/2 signalling in striatal neurons. Our findings identify the BET protein family, and Brd4 in particular, as novel regulators of basal and D1R-dependent transcription in rat striatal neurons, and delineate complex bi-directional effects of bromodomain inhibitors on neuronal transcription.

High-Content Single-Cell Förster Resonance Energy Transfer Imaging of Cultured Striatal Neurons Reveals Novel Cross-Talk in the Regulation of Nuclear Signaling by Protein Kinase A and Extracellular Signal-Regulated Kinase 1/2.

Genetically encoded biosensors can be used to track signaling events in living cells by measuring changes in fluorescence emitted by one or more fluorescent proteins. Here, we describe the use of genetically encoded biosensors based on Förster resonance energy transfer (FRET), combined with high-content microscopy, to image dynamic signaling events simultaneously in thousands of neurons in response to drug treatments. We first applied this approach to examine intercellular variation in signaling responses among cultured striatal neurons stimulated with multiple drugs. Using high-content FRET imaging and immunofluorescence, we identified neuronal subpopulations with unique responses to pharmacological manipulation and used nuclear morphology to identify medium spiny neurons within these heterogeneous striatal cultures. Focusing on protein kinase A (PKA) and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in the cytoplasm and nucleus, we noted pronounced intercellular differences among putative medium spiny neurons, in both the magnitude and kinetics of signaling responses to drug application. Importantly, a conventional "bulk" analysis that pooled all cells in culture yielded a different rank order of drug potency than that revealed by single-cell analysis. Using a single-cell analytical approach, we dissected the relative contributions of PKA and ERK1/2 signaling in striatal neurons and unexpectedly identified a novel role for ERK1/2 in promoting nuclear activation of PKA in striatal neurons. This finding adds a new dimension of signaling crosstalk between PKA and ERK1/2 with relevance to dopamine D1 receptor signaling in striatal neurons. In conclusion, high-content single-cell imaging can complement and extend traditional population-level analyses and provides a novel vantage point from which to study cellular signaling. SIGNIFICANCE STATEMENT: High-content imaging revealed substantial intercellular variation in the magnitude and pattern of intracellular signaling events driven by receptor stimulation. Since individual neurons within the same population can respond differently to a given agonist, interpreting measures of intracellular signaling derived from the averaged response of entire neuronal populations may not always reflect what happened at the single-cell level. This study uses this approach to identify a new form of cross-talk between PKA and ERK1/2 signaling in the nucleus of striatal neurons.

Lipidated peptides derived from intracellular loops 2 and 3 of the urotensin II receptor act as biased allosteric ligands.

Over the last decade, the urotensinergic system, composed of one G protein-coupled receptor and two endogenous ligands, has garnered significant attention as a promising new target for the treatment of various cardiovascular diseases. Indeed, this system is associated with various biomarkers of cardiovascular dysfunctions and is involved in changes in cardiac contractility, fibrosis, and hypertrophy contributing, like the angiotensinergic system, to the pathogenesis and progression of heart failure. Significant investment has been made toward the development of clinically relevant UT ligands for therapeutic intervention, but with little or no success to date. This system therefore remains to be therapeutically exploited. Pepducins and other lipidated peptides have been used as both mechanistic probes and potential therapeutics; therefore, pepducins derived from the human urotensin II receptor might represent unique tools to generate signaling bias and study hUT signaling networks. Two hUT-derived pepducins, derived from the second and the third intracellular loop of the receptor (hUT-Pep2 and [Trp1, Leu2]hUT-Pep3, respectively), were synthesized and pharmacologically characterized. Our results demonstrated that hUT-Pep2 and [Trp1, Leu2]hUT-Pep3 acted as biased ago-allosteric modulators, triggered ERK1/2 phosphorylation and, to a lesser extent, IP1 production, and stimulated cell proliferation yet were devoid of contractile activity. Interestingly, both hUT-derived pepducins were able to modulate human urotensin II (hUII)- and urotensin II-related peptide (URP)-mediated contraction albeit to different extents. These new derivatives represent unique tools to reveal the intricacies of hUT signaling and also a novel avenue for the design of allosteric ligands selectively targeting hUT signaling potentially.

New connections between ubiquitylation and methylation in the co-transcriptional histone modification network.

Co-transcriptional histone modifications are a ubiquitous feature of RNA polymerase II (RNAPII) transcription, with profound but incompletely understood effects on gene expression. Unlike the covalent marks found at promoters, which are thought to be instructive for transcriptional activation, these modifications occur in gene bodies as a result of transcription, which has made elucidation of their functions challenging. Here we review recent insights into the regulation and roles of two such modifications: monoubiquitylation of histone H2B at lysine 120 (H2Bub1) and methylation of histone H3 at lysine 36 (H3K36me). Both H2Bub1 and H3K36me are enriched in the coding regions of transcribed genes, with highly overlapping distributions, but they were thought to work largely independently. We highlight our recent demonstration that, as was previously shown for H3K36me, H2Bub1 signals to the histone deacetylase (HDAC) complex Rpd3S/Clr6-CII, and that Rpd3S/Clr6-CII and H2Bub1 function in the same pathway to repress aberrant antisense transcription initiating within gene coding regions. Moreover, both of these histone modification pathways are influenced by protein phosphorylation catalyzed by the cyclin-dependent kinases (CDKs) that regulate RNAPII elongation, chiefly Cdk9. Therefore, H2Bub1 and H3K36me are more tightly linked than previously thought, sharing both upstream regulatory inputs and downstream effectors. Moreover, these newfound connections suggest extensive, bidirectional signaling between RNAPII elongation complexes and chromatin-modifying enzymes, which helps to determine transcriptional outputs and should be a focus for future investigation.

Dopamine D1 receptor signalling in dyskinetic Parkinsonian rats revealed by fiber photometry using FRET-based biosensors.

As with many G protein-coupled receptors (GPCRs), the signalling pathways regulated by the dopamine D1 receptor (D1R) are dynamic, cell type-specific, and can change in the face of disease or drug exposures. In striatal neurons, the D1R activates cAMP/protein kinase A (PKA) signalling. However, in Parkinson's disease (PD), alterations in this pathway lead to functional upregulation of extracellular regulated kinases 1/2 (ERK1/2), contributing to L-DOPA-induced dyskinesia (LID). In order to detect D1R activation in vivo and to study the progressive dysregulation of D1R signalling in PD and LID, we developed ratiometric fiber-photometry with Förster resonance energy transfer (FRET) biosensors and optically detected PKA and ERK1/2 signalling in freely moving rats. We show that in Parkinsonian animals, D1R signalling through PKA and ERK1/2 is sensitized, but that following chronic treatment with L-DOPA, these pathways become partially desensitized while concurrently D1R activation leads to greater induction of dyskinesia.

Cdk9 and H2Bub1 signal to Clr6-CII/Rpd3S to suppress aberrant antisense transcription.

Mono-ubiquitylation of histone H2B (H2Bub1) and phosphorylation of elongation factor Spt5 by cyclin-dependent kinase 9 (Cdk9) occur during transcription by RNA polymerase II (RNAPII), and are mutually dependent in fission yeast. It remained unclear whether Cdk9 and H2Bub1 cooperate to regulate the expression of individual genes. Here, we show that Cdk9 inhibition or H2Bub1 loss induces intragenic antisense transcription of ∼10% of fission yeast genes, with each perturbation affecting largely distinct subsets; ablation of both pathways de-represses antisense transcription of over half the genome. H2Bub1 and phospho-Spt5 have similar genome-wide distributions; both modifications are enriched, and directly proportional to each other, in coding regions, and decrease abruptly around the cleavage and polyadenylation signal (CPS). Cdk9-dependence of antisense suppression at specific genes correlates with high H2Bub1 occupancy, and with promoter-proximal RNAPII pausing. Genetic interactions link Cdk9, H2Bub1 and the histone deacetylase Clr6-CII, while combined Cdk9 inhibition and H2Bub1 loss impair Clr6-CII recruitment to chromatin and lead to decreased occupancy and increased acetylation of histones within gene coding regions. These results uncover novel interactions between co-transcriptional histone modification pathways, which link regulation of RNAPII transcription elongation to suppression of aberrant initiation.

Signal profiling of the ß1AR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by ß1AR and ß2AR.

A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different ß-adrenergic ligands to engage five distinct signalling pathways downstream of the ß1-adrenergic receptor (ß1AR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the ßAR. These include coupling to Gz and G12 pathways. The signalling cascade linking the ß1AR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both Gs and G12 are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.

Therapeutic Targeting of the General RNA Polymerase II Transcription Machinery.

Inhibitors targeting the general RNA polymerase II (RNAPII) transcription machinery are candidate therapeutics in cancer and other complex diseases. Here, we review the molecular targets and mechanisms of action of these compounds, framing them within the steps of RNAPII transcription. We discuss the effects of transcription inhibitors in vitro and in cellular models (with an emphasis on cancer), as well as their efficacy in preclinical and clinical studies. We also discuss the rationale for inhibiting broadly acting transcriptional regulators or RNAPII itself in complex diseases.

Spt5 Phosphorylation and the Rtf1 Plus3 Domain Promote Rtf1 Function through Distinct Mechanisms.

Rtf1 is a conserved RNA polymerase II (RNAPII) elongation factor that promotes cotranscriptional histone modification, RNAPII transcript elongation, and mRNA processing. Rtf1 function requires the phosphorylation of Spt5, an essential RNAPII processivity factor. Spt5 is phosphorylated within its C-terminal domain (CTD) by cyclin-dependent kinase 9 (Cdk9), the catalytic component of positive transcription elongation factor b (P-TEFb). Rtf1 recognizes phosphorylated Spt5 (pSpt5) through its Plus3 domain. Since Spt5 is a unique target of Cdk9 and Rtf1 is the only known pSpt5-binding factor, the Plus3/pSpt5 interaction is thought to be a key Cdk9-dependent event regulating RNAPII elongation. Here, we dissect Rtf1 regulation by pSpt5 in the fission yeast Schizosaccharomyces pombe We demonstrate that the Plus3 domain of Rtf1 (Prf1 in S. pombe) and pSpt5 are functionally distinct and that they act in parallel to promote Prf1 function. This alternate Plus3 domain function involves an interface that overlaps the pSpt5-binding site and that can interact with single-stranded nucleic acid or with the polymerase-associated factor (PAF) complex in vitro We further show that the C-terminal region of Prf1, which also interacts with PAF, has a similar parallel function with pSpt5. Our results elucidate unexpected complexity underlying Cdk9-dependent pathways that regulate transcription elongation.

Differential Activation of P-TEFb Complexes in the Development of Cardiomyocyte Hypertrophy following Activation of Distinct G Protein-Coupled Receptors.

Pathological cardiac hypertrophy is driven by neurohormonal activation of specific G protein-coupled receptors (GPCRs) in cardiomyocytes and is accompanied by large-scale changes in cardiomyocyte gene expression. These transcriptional changes require activity of positive transcription elongation factor b (P-TEFb), which is recruited to target genes by the bromodomain protein Brd4 or the super elongation complex (SEC). Here, we describe GPCR-specific regulation of these P-TEFb complexes and a novel mechanism for activating Brd4 in primary neonatal rat cardiomyocytes. The SEC was required for the hypertrophic response downstream of either the α1-adrenergic receptor (α1-AR) or the endothelin receptor (ETR). In contrast, Brd4 inhibition selectively impaired the α1-AR response. This was corroborated by the finding that the activation of α1-AR, but not ETR, increased Brd4 occupancy at promoters and superenhancers of hypertrophic genes. Transcriptome analysis demonstrated that the activation of both receptors initiated similar gene expression programs, but that Brd4 inhibition attenuated hypertrophic genes more robustly following α1-AR activation. Finally, we show that protein kinase A (PKA) is required for α1-AR stimulation of Brd4 chromatin occupancy. The differential role of the Brd4/P-TEFb complex in response to distinct GPCR pathways has potential clinical implications, as therapies targeting this complex are currently being explored for heart failure.

Histone H2B Ubiquitylation Regulates Histone Gene Expression by Suppressing Antisense Transcription in Fission Yeast.

Histone H2B monoubiquitylation (H2Bub1) is tightly linked to RNA polymerase II transcription elongation, and is also directly implicated in DNA replication and repair. Loss of H2Bub1 is associated with defects in cell cycle progression, but how these are related to its various functions, and the underlying mechanisms involved, is not understood. Here we describe a role for H2Bub1 in the regulation of replication-dependent histone genes in the fission yeast Schizosaccharomyces pombe H2Bub1 activates histone genes indirectly by suppressing antisense transcription of ams2+ -a gene encoding a GATA-type transcription factor that activates histone genes and is required for assembly of centromeric chromatin. Mutants lacking the ubiquitylation site in H2B or the H2B-specific E3 ubiquitin ligase Brl2 had elevated levels of ams2+ antisense transcripts and reduced Ams2 protein levels. These defects were reversed upon inhibition of Cdk9-an ortholog of the kinase component of positive transcription elongation factor b (P-TEFb)-indicating that they likely resulted from aberrant transcription elongation. Reduced Cdk9 activity also partially rescued chromosome segregation phenotypes of H2Bub1 mutants. In a genome-wide analysis, loss of H2Bub1 led to increased antisense transcripts at over 500 protein-coding genes in H2Bub1 mutants; for a subset of these, including several genes involved in chromosome segregation and chromatin assembly, antisense derepression was Cdk9-dependent. Our results highlight antisense suppression as a key feature of cell cycle-dependent gene regulation by H2Bub1, and suggest that aberrant transcription elongation may underlie the effects of H2Bub1 loss on cell cycle progression.

A Cdk9-PP1 switch regulates the elongation-termination transition of RNA polymerase II.

The end of the RNA polymerase II (Pol II) transcription cycle is strictly regulated to prevent interference between neighbouring genes and to safeguard transcriptome integrity 1 . The accumulation of Pol II downstream of the cleavage and polyadenylation signal can facilitate the recruitment of factors involved in mRNA 3'-end formation and termination 2 , but how this sequence is initiated remains unclear. In a chemical-genetic screen, human protein phosphatase 1 (PP1) isoforms were identified as substrates of positive transcription elongation factor b (P-TEFb), also known as the cyclin-dependent kinase 9 (Cdk9)-cyclin T1 (CycT1) complex 3 . Here we show that Cdk9 and PP1 govern phosphorylation of the conserved elongation factor Spt5 in the fission yeast Schizosaccharomyces pombe. Cdk9 phosphorylates both Spt5 and a negative regulatory site on the PP1 isoform Dis2 4 . Sites targeted by Cdk9 in the Spt5 carboxy-terminal domain can be dephosphorylated by Dis2 in vitro, and dis2 mutations retard Spt5 dephosphorylation after inhibition of Cdk9 in vivo. Chromatin immunoprecipitation and sequencing analysis indicates that Spt5 is dephosphorylated as transcription complexes traverse the cleavage and polyadenylation signal, concomitant with the accumulation of Pol II phosphorylated at residue Ser2 of the carboxy-terminal domain consensus heptad repeat 5 . A conditionally lethal Dis2-inactivating mutation attenuates the drop in Spt5 phosphorylation on chromatin, promotes transcription beyond the normal termination zone (as detected by precision run-on transcription and sequencing 6 ) and is genetically suppressed by the ablation of Cdk9 target sites in Spt5. These results suggest that the transition of Pol II from elongation to termination coincides with a Dis2-dependent reversal of Cdk9 signalling-a switch that is analogous to a Cdk1-PP1 circuit that controls mitotic progression 4 .

Receptor- and cellular compartment-specific activation of the cAMP/PKA pathway by α1-adrenergic and ETA endothelin receptors.

The signalling functions of many G protein-coupled receptors (GPCRs) expressed in the myocardium are incompletely understood. Among these are the endothelin receptor (ETR) family and α1-adrenergic receptor (α1-AR), which are thought to couple to the G protein Gαq. In this study, we used transcriptome analysis to compare the signalling networks downstream of these receptors in primary neonatal rat cardiomyocytes. This analysis indicated increased expression of target genes of cAMP responsive element modulator (CREM) after 24 h treatment with the α1-AR agonist phenylephrine, but not the ETR agonist endothelin-1, suggesting a specific role for the α1-AR in promoting cAMP production in cardiomyocytes. To validate the difference observed between these two GPCRs, we used heterologous expression of the receptors and genetically encoded biosensors in HEK 293 cell lines. We validated that both α1A- and α1B-AR subtypes were able to lead to the accumulation of cAMP in response to phenylephrine in both the nucleus and cytoplasm in a Gαs-dependent manner. However, the ETR subtype ETA did not affect cAMP levels in either compartment. All three receptors were coupled to Gαq signalling as expected. Further, we showed that activation of PKA in different compartments was α1-AR subtype specific, with α1B-AR able to activate PKA in the cytoplasm and nucleus and α1A-AR only able to in the nucleus. We provide evidence for a pathway downstream of the α1-AR, and show that distinct pools of a receptor lead to differential activation of downstream effector proteins dependent on their cellular compartment.

Chromatin Immunoprecipitation of Histone Modifications in Fission Yeast.

Chromatin immunoprecipitation (ChIP) is a sensitive, accurate, and reliable technique widely used to analyze protein-DNA interactions at specific binding sites in vivo. It has been a particularly powerful technique for mapping of histone modification patterns both at individual loci and genome-wide. Here we provide a detailed protocol for ChIP of histone modifications associated with active transcription in fission yeast (Schizosaccharomyces pombe).

Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification.

Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast Schizosaccharomyces pombe. Steady-state levels of methylation at histone H3 lysines 4 (H3K4me) and 36 (H3K36me) were sensitive to multiple mutations of the Rpb1 CTD repeat motif (Y1S2P3T4S5P6S7). Ablation of the Spt5 CTD phospho-site Thr1 reduced H3K4me levels but had minimal effects on H3K36me. Nonetheless, Spt5 CTD mutations potentiated the effects of Rpb1 CTD mutations on H3K36me, suggesting overlapping functions. Phosphorylation of Rpb1 Ser2 by the Cdk12 orthologue Lsk1 positively regulated H3K36me but negatively regulated H3K4me. H3K36me and histone H2B monoubiquitylation required Rpb1 Ser5 but were maintained upon inactivation of Mcs6/Cdk7, the major kinase for Rpb1 Ser5 in vivo, implicating another Ser5 kinase in these regulatory pathways. Our results elaborate the CTD 'code' for co-transcriptional histone modifications.