Phosphorylation Of Carboxyl-terminal Domain Research Articles

Regulation of mature mRNA levels by RNA processing efficiency.

Transcription and co-transcriptional processes, including pre-mRNA splicing and mRNA cleavage and polyadenylation, regulate the production of mature mRNAs. The carboxyl terminal domain (CTD) of RNA polymerase (pol) II, which comprises 52 repeats of the Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 peptide, is involved in the coordination of transcription with co-transcriptional processes. The pol II CTD is dynamically modified by protein phosphorylation, which regulates recruitment of transcription and co-transcriptional factors. We have investigated whether mature mRNA levels from intron-containing protein-coding genes are related to pol II CTD phosphorylation, RNA stability, and pre-mRNA splicing and mRNA cleavage and polyadenylation efficiency. We find that genes that produce a low level of mature mRNAs are associated with relatively high phosphorylation of the pol II CTD Thr4 residue, poor RNA processing, increased chromatin association of transcripts, and shorter RNA half-life. While these poorly-processed transcripts are degraded by the nuclear RNA exosome, our results indicate that in addition to RNA half-life, chromatin association due to a low RNA processing efficiency also plays an important role in the regulation of mature mRNA levels.

Evaluating Spatiotemporal Dynamics of Phosphorylation of RNA Polymerase II Carboxy-Terminal Domain by Ultraviolet Photodissociation Mass Spectrometry.

The critical role of site-specific phosphorylation in eukaryotic transcription has motivated efforts to decipher the complex phosphorylation patterns exhibited by the carboxyl-terminal domain (CTD) of RNA polymerase II. Phosphorylation remains a challenging post-translational modification to characterize by mass spectrometry owing to the labile phosphate ester linkage and low stoichiometric prevalence, two features that complicate analysis by high-throughput MS/MS methods. Identifying phosphorylation sites represents one significant hurdle in decrypting the CTD phosphorylation, a problem exaggerated by a large number of potential phosphorylation sites. An even greater obstacle is decoding the dynamic phosphorylation pattern along the length of the periodic CTD sequence. Ultraviolet photodissociation (UVPD) is a high-energy ion activation method that provides ample backbone cleavages of peptides while preserving labile post-translational modifications that facilitate their confident localization. Herein, we report a quantitative parallel reaction monitoring (PRM) method developed to monitor spatiotemporal changes in site-specific Ser5 phosphorylation of the CTD by cyclin-dependent kinase 7 (CDK7) using UVPD for sequence identification, phosphosite localization, and differentiation of phosphopeptide isomers. We capitalize on the series of phospho-retaining fragment ions produced by UVPD to create unique transition lists that are pivotal for distinguishing the array of phosphopeptides generated from the CTD.

A novel RNA pol II CTD interaction site on the mRNA capping enzyme is essential for its allosteric activation.

Recruitment of the mRNA capping enzyme (CE/RNGTT) to the site of transcription is essential for the formation of the 5′ mRNA cap, which in turn ensures efficient transcription, splicing, polyadenylation, nuclear export and translation of mRNA in eukaryotic cells. The CE GTase is recruited and activated by the Serine-5 phosphorylated carboxyl-terminal domain (CTD) of RNA polymerase II. Through the use of molecular dynamics simulations and enhanced sampling techniques, we provide a systematic and detailed characterization of the human CE–CTD interface, describing the effect of the CTD phosphorylation state, length and orientation on this interaction. Our computational analyses identify novel CTD interaction sites on the human CE GTase surface and quantify their relative contributions to CTD binding. We also identify, for the first time, allosteric connections between the CE GTase active site and the CTD binding sites, allowing us to propose a mechanism for allosteric activation. Through binding and activity assays we validate the novel CTD binding sites and show that the CDS2 site is essential for CE GTase activity stimulation. Comparison of the novel sites with cocrystal structures of the CE–CTD complex in different eukaryotic taxa reveals that this interface is considerably more conserved than previous structures have indicated.

CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation.

Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.

Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates.

Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive carboxyl-terminal domain (CTD) of its largest subunit, RPB1, at specific residues. A variety of techniques exist to identify and quantify the degree of CTD phosphorylation, including phosphorylation-specific antibodies and mass spectrometry. Electrophoretic mobility shift assays (EMSAs) have been utilized since the discovery of CTD phosphorylation and continue to represent a simple, direct, and widely applicable approach for qualitatively monitoring CTD phosphorylation. We present a standardized method for EMSA analysis of recombinant GST-CTD substrates phosphorylated by a variety of CTD kinases. Strategies to analyze samples under both denatured/reduced and semi-native conditions are provided. This method represents a simple, direct, and reproducible means to monitor CTD phosphorylation in recombinant substrates utilizing equipment common to molecular biology labs and readily applicable to downstream analyses including immunoblotting and mass spectrometry.

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

CTD-dependent and -independent mechanisms govern co-transcriptional capping of Pol II transcripts

Co-transcriptional capping of RNA polymerase II (Pol II) transcripts by capping enzyme proceeds orders of magnitude more efficiently than capping of free RNA. Previous studies brought to light a role for the phosphorylated Pol II carboxyl-terminal domain (CTD) in activation of co-transcriptional capping; however, CTD phosphorylation alone could not account for the observed magnitude of activation. Here, we exploit a defined Pol II transcription system that supports both CTD phosphorylation and robust activation of capping to dissect the mechanism of co-transcriptional capping. Taken together, our findings identify a CTD-independent, but Pol II-mediated, mechanism that functions in parallel with CTD-dependent processes to ensure optimal capping, and they support a “tethering” model for the mechanism of activation.

Widespread and precise reprogramming of yeast protein-genome interactions in response to heat shock.

Gene expression is controlled by a variety of proteins that interact with the genome. Their precise organization and mechanism of action at every promoter remains to be worked out. To better understand the physical interplay among genome-interacting proteins, we examined the temporal binding of a functionally diverse subset of these proteins: nucleosomes (H3), H2AZ (Htz1), SWR (Swr1), RSC (Rsc1, Rsc3, Rsc58, Rsc6, Rsc9, Sth1), SAGA (Spt3, Spt7, Ubp8, Sgf11), Hsf1, TFIID (Spt15/TBP and Taf1), TFIIB (Sua7), TFIIH (Ssl2), FACT (Spt16), Pol II (Rpb3), and Pol II carboxyl-terminal domain (CTD) phosphorylation at serines 2, 5, and 7. They were examined under normal and acute heat shock conditions, using the ultrahigh resolution genome-wide ChIP-exo assay in Saccharomyces cerevisiae. Our findings reveal a precise positional organization of proteins bound at most genes, some of which rapidly reorganize within minutes of heat shock. This includes more precise positional transitions of Pol II CTD phosphorylation along the 5′ ends of genes than previously seen. Reorganization upon heat shock includes colocalization of SAGA with promoter-bound Hsf1, a change in RSC subunit enrichment from gene bodies to promoters, and Pol II accumulation within promoter/+1 nucleosome regions. Most of these events are widespread and not necessarily coupled to changes in gene expression. Together, these findings reveal protein–genome interactions that are robustly reprogrammed in precise and uniform ways far beyond what is elicited by changes in gene expression.

Profiling RNA Polymerase II Phosphorylation Genome-Wide in Fission Yeast.

The RNA polymerase II carboxyl-terminal domain (CTD) consists of tandem repeats of consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Dynamic posttranslational modifications of the CTD generate a CTD code crucial for the cotranscriptional recruitment of factors that control transcription, chromatin modification, and RNA processing. Analysis of CTD phosphorylation by ChIP (Chromatin ImmunoPrecipitation) coupled with high-throughput DNA sequencing (ChIP-seq) is a powerful tool to investigate the changes in CTD phosphorylation during the transcription cycle. In this chapter, we describe a ChIP-seq protocol to profile the different CTD phospho-marks in fission yeast. Using this protocol, we have found that Tyr1P, Ser2P, and Thr4P signals are highest at gene 3' ends, whereas Ser5P is enriched across the gene bodies.

Promoter-Terminator Gene Loops Affect Alternative 3′-End Processing in Yeast

Many eukaryotic genes undergo alternative 3'-end poly(A)-site selection producing transcript isoforms with 3'-UTRs of different lengths and post-transcriptional fates. Gene loops are dynamic structures that juxtapose the 3'-ends of genes with their promoters. Several functions have been attributed to looping, including memory of recent transcriptional activity and polarity of transcription initiation. In this study, we investigated the relationship between gene loops and alternative poly(A)-site. Using the KlCYC1 gene of the yeast Kluyveromyces lactis, which includes a single promoter and two poly(A) sites separated by 394 nucleotides, we demonstrate in two yeast species the formation of alternative gene loops (L1 and L2) that juxtapose the KlCYC1 promoter with either proximal or distal 3'-end processing sites, resulting in the synthesis of short and long forms of KlCYC1 mRNA. Furthermore, synthesis of short and long mRNAs and formation of the L1 and L2 loops are growth phase-dependent. Chromatin immunoprecipitation experiments revealed that the Ssu72 RNA polymerase II carboxyl-terminal domain phosphatase, a critical determinant of looping, peaks in early log phase at the proximal poly(A) site, but as growth phase advances, it extends to the distal site. These results define a cause-and-effect relationship between gene loops and alternative poly(A) site selection that responds to different physiological signals manifested by RNA polymerase II carboxyl-terminal domain phosphorylation status.

Punctuation and syntax of the RNA polymerase II CTD code in fission yeast

The primary structure and phosphorylation pattern of the tandem Y(1)S(2)P(3)T(4)S(5)P(6)S(7) repeats of the RNA polymerase II carboxyl-terminal domain (CTD) convey information about the transcription apparatus--a CTD code--to a large ensemble of CTD-binding receptor proteins. Four of the seven coding "letters" of the fission yeast CTD (Tyr1, Pro3, Ser5, Pro6) are essential in vivo, but the grammatical rules of the code are obscure. Here we show that the minimal fission yeast CTD coding unit is a decapeptide Y(1)S(2)P(3)T(4)S(5)P(6)S(7)Y(1)S(2)P(3) and the spacing between coding units is flexible; the coding unit must contain two Tyr1 residues and the spacing between consecutive tyrosines is important; Ser5-PO(4)-Pro6 comprises an essential two-letter code "word" that is read by the mRNA capping apparatus; and a threshold number of Ser5-PO(4)-Pro6 words are needed to comprise a readable "sentence" of CTD information. Bypassing the essentiality of the Ser5 and Pro6 letters by fusion of capping enzymes to the CTD helped reveal how CTD phosphorylation circuits are wired in vivo. We found that the Ser2-PO(4) mark is independent of Ser5, Pro6, Ser7, and Thr4, whereas the Ser5-PO(4) mark is independent of Ser2, Ser7, and Thr4. These results provide unique insights to the reading and writing of the CTD code.

Architecture of the Mediator Head Module

Mediator is a key regulator of eukaryotic transcription, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II). In the yeast S. cerevisiae, Mediator is organized into three modules (Head, Middle/Arm and Tail) that together comprise 21 subunits with a total mass over 1 MDa. Our understanding of Mediator assembly, and of its role in regulating transcription, has so far been hampered by limited structural information. We solved the crystal structure of the Mediator Head module (7 subunits, 223 kDa) at 4.3 angstrom resolution, revealing three distinct domains (Fixed jaw, Movable jaw, and Neck) that come together to form a flexible central joint region. Strikingly, we discovered a novel structure in the Neck domain, in which a total of ten helices from five different subunits form a large multi‐helical bundle as a single structural unit. This unique helical bundle structure ensures stable assembly of the Head subunits and provides binding sites for general transcription factors and Pol II. Mapping previously identified genetic mutations onto our Head structure suggests that the Head module may juxtapose TFIIH and the carboxyl‐terminal domain (CTD) of the largest subunit of Pol II (Rpb1) to facilitate CTD phosphorylation. These results reveal architectural principles underlying the role of Mediator in the regulation of gene expression. Supported by NSF, AHA and NIH

Flavopiridol induces phosphorylation of AKT in a human glioblastoma cell line, in contrast to siRNA-mediated silencing of Cdk9: Implications for drug design and development

Cdk9 and Cdk7 are cdc2-like serine/threonine kinases that stabilize RNA transcript elongation through RNA polII carboxyl terminal domain (CTD) phosphorylation and are considered suitable targets for cancer therapy. The effects of flavopiridol and of siRNA-mediated inhibition of Cdk9 and/or Cdk7 were analyzed in human glioblastoma and human prostate cancer cell lines. One finding revealed that Cdk9 and Cdk7 could substitute each other in RNA polII CTD phosphorylation in contrast to the in vitro system. Thus, a simultaneous inhibition of Cdk9 and Cdk7 might be required both for targeting malignant cells and developing a platform for microarray analysis. However, these two pathways are not redundant, as indicated by differential effects observed in cell cycle regulation following siRNA-mediated inhibition of Cdk9 and/or Cdk7 in human PC3 prostate cancer cell line. Specifically, siRNA-mediated inhibition of Cdk9 caused a shift from G0/G1 to G2/M phase in human PC3 prostate cancer cell line. Another finding showed that flavopiridol treatment induced a substantial AKT-Ser473 phosphorylation in human glioblastoma T98G cell line in contrast to siRNA-mediated inhibition of Cdk9 and Cdk9 combined with Cdk7, whereas siRNA-mediated silencing of Cdk7 caused a minor increase in AKT-Ser473 phosphorylation. AKT-Ser473 is a hallmark of AKT pathway activation and may protect cells from apoptosis. This finding also shows that Cdk9 and Cdk7 pathways are not redundant and may have important implications in drug development and for studying the mechanism of chemoresistance in malignant cells.

Architecture of the Mediator Head Module

Mediator is a key regulator of eukaryotic transcription, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II). In the yeast S. cerevisiae, Mediator is organized into three modules (Head, Middle/Arm and Tail) that together comprise 21 subunits with a total mass over 1 MDa. Our understanding of Mediator assembly, and of its role in regulating transcription, has so far been hampered by limited structural information. By using state-of-the-art protein complex engineering and phasing methods, we solved the structure of the Mediator Head module (7 subunits, 223 kDa) at 4.3 angstrom resolution. Our structure reveals three distinct domains (Fixed jaw, Movable jaw, and Neck) that come together to form a flexible central joint region. Strikingly, we discovered a novel and elaborate structure in the Neck domain, in which a total of ten helices from five different subunits form a large multi-helical bundle as a single structure unit. An intricate pattern of interactions within this helical bundle ensures stable assembly of the Head subunits and provides binding sites for general transcription factors (GTFs) and Pol II. Mapping previously identified genetic mutations onto our Head module X-ray structure suggests that the Head module may juxtapose TFIIH and the carboxyl-terminal domain (CTD) of the largest subunit of Pol II (Rpb1) to facilitate CTD phosphorylation. These results reveal architectural principles underlying the role of Mediator in the regulation of gene expression.

RNA Polymerase II Carboxyl-terminal Domain Phosphorylation Regulates Protein Stability of the Set2 Methyltransferase and Histone H3 Di- and Trimethylation at Lysine 36

RNA Polymerase II Carboxyl-terminal Domain Phosphorylation Regulates Protein Stability of the Set2 Methyltransferase and Histone H3 Di- and Trimethylation at Lysine 36

RNA Polymerase II Carboxyl-terminal Domain Phosphorylation Regulates Protein Stability of the Set2 Methyltransferase and Histone H3 Di- and Trimethylation at Lysine 36

Methylation of lysine 36 on histone H3 (H3K36) is catalyzed by the Set2 methyltransferase and is linked to transcriptional regulation. Previous studies have shown that trimethylation of H3K36 by Set2 is directed through its association with the phosphorylated repeats of the RNA polymerase C-terminal domain (RNAPII CTD). Here, we show that disruption of this interaction through the use of yeast mutants defective in CTD phosphorylation at serine 2 results in a destabilization of Set2 protein levels and H3K36 methylation. Consistent with this, we find that Set2 has a short half-life and is co-regulated, with RNAPII CTD phosphorylation levels, during logarithmic growth in yeast. To probe the functional consequence of uncoupling Set2-RNAPII regulation, we expressed a truncated and more stable form of Set2 that is capable of dimethylation but not trimethylation in vivo. Results of high throughput synthetic genetic analyses show that this Set2 variant has distinct genetics from either SET2 or set2Δ and is synthetically sick or lethal with a number of transcription elongation mutants. Collectively, these results provide molecular insight into the regulation of Set2 protein levels that influence H3K36 methylation states.

The Eleven-Nineteen Lysine-rich Leukemia Gene (ELL2) Influences the Histone H3 Protein Modifications Accompanying the Shift to Secretory Immunoglobulin Heavy Chain mRNA Production

In plasma cells, immunoglobulin heavy chain (IgH) secretory-specific mRNA is made in high abundance as a result of both increased promoter proximal poly(A) site choice and weak splice-site skipping. Ell2, the eleven-nineteen lysine rich leukemia gene, is a transcription elongation factor that is induced ∼6-fold in plasma cells and has been shown to drive secretory-specific mRNA production. Reducing ELL2 by siRNA, which reduced processing to the secretion-specific poly(A) site, also influenced the methylations of histone H3K4 and H3K79 on the IgH gene and impacted positive transcription factor b (pTEFb), Ser-2 carboxyl-terminal phosphorylation, and polyadenylation factor additions to RNA polymerase II. The multiple lineage leukemia gene (MLL) and Dot1L associations with the IgH gene were also impaired in the absence of ELL2. To investigate the link between histone modifications, transcription elongation, and alternative RNA processing in IgH mRNA production, we performed chromatin immunoprecipitation on cultured mouse B and plasma cells bearing the identical IgH γ2a gene. In the plasma cells, as compared with the B cells, the H3K4 and H3K79 methylations extended farther downstream, past the IgH enhancer to the end of the transcribed region. Thus the downstream H3K4 and H3K79 methylation of the IgH associated chromatin in plasma cells is associated with increased polyadenylation and exon skipping, resulting from the actions of ELL2 transcription elongation factor.

Cell death induction in resting lymphocytes by pan-Cdk inhibitor, but not by Cdk4/6 selective inhibitor

Immunosuppression is one of the common side effects of many anti-tumor agents targeting proliferating cells. We previously reported the development of a new class of pan-cyclin-dependent kinase (Cdk) inhibitor compounds that induce immunosuppression in rodents. Here, we demonstrated that a pan-Cdk inhibitor, Compound 1 very rapidly reduced white blood cells in mice, only 8h after administration. Compound 1 induced death of peripheral blood cells or purified resting (non-stimulated) lymphocytes ex vivo. Cell death was induced very rapidly, after 4h of incubation, suggesting that acute immunosuppression observed in rodents might be, at least in part, due to direct cytotoxic effects of Compound 1 on resting lymphocytes. While cell cycle-related Cdks were not activated, the carboxyl terminal domain (CTD) of the largest subunit of RNA polymerase II was phosphorylated, indicating activation of Cdk7 or Cdk9, which phosphorylates this domain, in resting lymphocytes. Indeed, the pan-Cdk inhibitor suppressed CTD phosphorylation in resting cells at the dose required for cell death induction. Inhibition of Cdk7 or Cdk9 by Compound 1 was also confirmed by suppression of nuclear factor-kappa B (NF-κB)-dependent transcription activity in the human cancer cell line U2OS. Interestingly, a Cdk4/6 inhibitor with selectivity against Cdk7 and Cdk9 did not induce cell death in resting lymphocytes. These results suggest that CTD phosphorylation possibly by Cdk7 or Cdk9 might be important for survival of resting lymphocytes and that Cdk inhibitors without inhibitory activity on these kinases might be an attractive agent for cancer chemotherapy.

Separable Functions of the Fission Yeast Spt5 Carboxyl-Terminal Domain (CTD) in Capping Enzyme Binding and Transcription Elongation Overlap with Those of the RNA Polymerase II CTD

An interaction network connecting mRNA capping enzymes, the RNA polymerase II (Pol II) carboxyl-terminal domain (CTD), elongation factor Spt5, and the Cdk7 and Cdk9 protein kinases is thought to comprise a transcription elongation checkpoint. A crux of this network is Spt5, which regulates early transcription elongation and has an imputed role in pre-mRNA processing via its physical association with capping enzymes. Schizosaccharomyces pombe Spt5 has a distinctive CTD composed of tandem nonapeptide repeats of the consensus sequence (1)TPAWNSGSK(9). The Spt5 CTD binds the capping enzymes and is a substrate for threonine phosphorylation by the Cdk9 kinase. Here we report that deletion of the S. pombe Spt5 CTD results in slow growth and aberrant cell morphology. The severity of the spt5-DeltaCTD phenotype is exacerbated by truncation of the Pol II CTD and ameliorated by overexpression of the capping enzymes RNA triphosphatase and RNA guanylyltransferase. These results suggest that the Spt5 and Pol II CTDs play functionally overlapping roles in capping enzyme recruitment. We probed structure-activity relations of the Spt5 CTD by alanine scanning of the consensus nonapeptide. The T1A change abolished CTD phosphorylation by Cdk9 but did not affect CTD binding to the capping enzymes. The T1A and P2A mutations elicited cold-sensitive (cs) and temperature-sensitive (ts) growth defects and conferred sensitivity to growth inhibition by 6-azauracil that was exacerbated by partial truncations of the Pol II CTD. The T1A phenotypes were rescued by a phosphomimetic T1E change but not by capping enzyme overexpression. These results imply a positive role for Spt5 CTD phosphorylation in Pol Il transcription elongation in fission yeast, distinct from its capping enzyme interactions. Viability of yeast cells bearing both Spt5 CTD T1A and Pol II CTD S2A mutations heralds that the Cdk9 kinase has an essential target other than Spt5 and Pol II CTD-Ser2.

Positive Transcription Elongation Factor b Activity in Compensatory Myocardial Hypertrophy is Regulated by Cardiac Lineage Protein-1

Emerging evidence illustrates the importance of the positive transcription elongation factor (P-TEF)b in control of global RNA synthesis, which constitutes a major feature of the compensatory response to diverse hypertrophic stimuli in cardiomyocytes. P-TEFb complex, composed of cyclin T and cdk9, is critical for elongation of nascent RNA chains via phosphorylation of the carboxyl-terminal domain of RNA polymerase (Pol) II. We and others have shown that the activity of P-TEFb is inhibited by its association with cardiac lineage protein (CLP)-1, the mouse homolog of human HEXIM1, in various physiological and pathological conditions. To investigate the mechanism of control of P-TEFb activity by CLP-1 in cardiac hypertrophy, we used a transgenic mouse model of hypertrophy caused by overexpression of calcineurin in the heart. We observed that the level of CLP-1 associated with P-TEFb was reduced markedly in hypertrophic hearts. We also generated bigenic mice (MHC-cyclin T1/CLP-1(+/-)) by crossing MHC-cyclin T1 transgenic mice with CLP-1 heterozygote. The bigenic mice exhibit enhanced susceptibility to hypertrophy that is accompanied with an increase in cdk9 activity via an increase in serine 2 phosphorylation of carboxyl-terminal domain and an increase in GLUT1/GLUT4 ratio. These mice have compensated systolic function without evidence of fibrosis and reduced lifespan. These data suggest that the reduced level of CLP-1 introduced in the background of elevated levels of cyclin T1 elevates derepression of P-TEFb activity and emphasizes the importance of the role of CLP-1 in the mechanism governing compensatory hypertrophy in cardiomyocytes.