Updating the CTD Story: From Tail to Epic

Eukaryotic RNA polymerase II (RNAPII) not only synthesizes mRNA but also coordinates transcription-related processes via its unique C-terminal repeat domain (CTD). The CTD is an RNAPII-specific protein segment consisting of repeating heptads with the consensus sequence Y1S2P3T4S5P6S7 that has been shown to be extensively post-transcriptionally modified in a coordinated, but complicated, manner. Recent discoveries of new modifications, kinases, and binding proteins have challenged previously established paradigms. In this paper, we examine results and implications of recent studies related to modifications of the CTD and the respective enzymes; we also survey characterizations of new CTD-binding proteins and their associated processes and new information regarding known CTD-binding proteins. Finally, we bring into focus new results that identify two additional CTD-associated processes: nucleocytoplasmic transport of mRNA and DNA damage and repair

Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12–cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.United States. National Institutes of Health (HG002668)United States. National Institutes of Health (CA109901

Covalent targeting of remote cysteine residues to develop CDK12 and CDK13 inhibitors

Cyclin-dependent kinases 12 and 13 (CDK12 and CDK13) play critical roles in the regulation of gene transcription. However, the absence of CDK12 and CDK13 inhibitors has hindered the ability to investigate the consequences of their inhibition in healthy cells and cancer cells. Here we describe the rational design of a first-in-class CDK12 and CDK13 covalent inhibitor, THZ531. Co-crystallization of THZ531 with CDK12–cyclin K indicates that THZ531 irreversibly targets a cysteine located outside the kinase domain. THZ531 causes a loss of gene expression with concurrent loss of elongating and hyperphosphorylated RNA polymerase II. In particular, THZ531 substantially decreases the expression of DNA damage response genes and key super-enhancer-associated transcription factor genes. Coincident with transcriptional perturbation, THZ531 dramatically induced apoptotic cell death. Small molecules capable of specifically targeting CDK12 and CDK13 may thus help identify cancer subtypes that are particularly dependent on their kinase activities.United States. National Institutes of Health (HG002668)United States. National Institutes of Health (CA109901

CDK12 Activity-Dependent Phosphorylation Events in Human Cells

We asked whether the C-terminal repeat domain (CTD) kinase, CDK12/CyclinK, phosphorylates substrates in addition to the CTD of RPB1, using our CDK12analog-sensitive HeLa cell line to investigate CDK12 activity-dependent phosphorylation events in human cells. Characterizing the phospho-proteome before and after selective inhibition of CDK12 activity by the analog 1-NM-PP1, we identified 5,644 distinct phospho-peptides, among which were 50 whose average relative amount decreased more than 2-fold after 30 min of inhibition (none of these derived from RPB1). Half of the phospho-peptides actually showed >3-fold decreases, and a dozen showed decreases of 5-fold or more. As might be expected, the 40 proteins that gave rise to the 50 affected phospho-peptides mostly function in processes that have been linked to CDK12, such as transcription and RNA processing. However, the results also suggest roles for CDK12 in other events, notably mRNA nuclear export, cell differentiation and mitosis. While a number of the more-affected sites resemble the CTD in amino acid sequence and are likely direct CDK12 substrates, other highly-affected sites are not CTD-like, and their decreased phosphorylation may be a secondary (downstream) effect of CDK12 inhibition

A DNA Damage Response System Associated with the phosphoCTD of Elongating RNA Polymerase II

<div><p>RNA polymerase II translocates across much of the genome and since it can be blocked by many kinds of DNA lesions, detects DNA damage proficiently; it thereby contributes to DNA repair and to normal levels of DNA damage resistance. However, the components and mechanisms that respond to polymerase blockage are largely unknown, except in the case of UV-induced damage that is corrected by nucleotide excision repair. Because elongating RNAPII carries with it numerous proteins that bind to its hyperphosphorylated CTD, we tested for effects of interfering with this binding. We find that expressing a decoy CTD-carrying protein in the nucleus, but not in the cytoplasm, leads to reduced DNA damage resistance. Likewise, inducing aberrant phosphorylation of the CTD, by deleting <i>CTK1</i>, reduces damage resistance and also alters rates of homologous recombination-mediated repair. In line with these results, extant data sets reveal a remarkable, highly significant overlap between phosphoCTD-associating protein genes and DNA damage-resistance genes. For one well-known phosphoCTD-associating protein, the histone methyltransferase Set2, we demonstrate a role in DNA damage resistance, and we show that this role requires the phosphoCTD binding ability of Set2; surprisingly, Set2’s role in damage resistance does not depend on its catalytic activity. To explain all of these observations, we posit the existence of a <u>C</u>TD-<u>A</u>ssociated DNA damage <u>R</u>esponse (CAR) system, organized around the phosphoCTD of elongating RNAPII and comprising a subset of phosphoCTD-associating proteins.</p></div

PhosphoCTD-associating proteins (PCAPs) were identified by Phatnani et al. [<b>20</b>] and assigned to likely functional categories.

<p> Proteins in red (CAR proteins) are products of genes identified by Bennett and colleagues as required for normal resistance to ionizing radiation (IR) (23,24) or doxorubicin (DX) (26). Bold = non-essential; light = essential; <u>underlined</u> = <u>binds directly to PCTD</u>; <i>italics = does not bind directly to PCTD</i>.</p

Damage resistance function of Set2 requires phosphoCTD binding but not catalytic activity.

<p><i>(A)</i> Primary structure of Set2 showing SET (catalytic) domain and SRI (CTD binding) domain. Position of catalytic point mutations is also illustrated. <i>(B)</i> The SRI domain of Set2 is required for damage resistance. Serial dilutions of <i>SET2 WT</i>, Δ<i>SRI</i> and complete gene deletion (<i>set2</i>Δ) strains were spotted on rich (YPD) medium containing either zero or 0.02% MMS, grown for 3 days at 30°C and photographed. “No MMS” results show that very similar numbers of cells were spotted for the three strains and that growth rates are quite similar (size of isolated colonies). All strains were affected by 0.02% MMS, for both survival and growth rate, but relative to WT (“normal” level of resistance) <i>set2</i>Δ and Δ<i>SRI</i> were less resistant. <i>(C)</i> Damage resistance does not depend on histone methyltransferase activity of Set2. Five-fold serial dilutions of <i>set2</i>Δ strains covered by various mutants of Set2 were plated with or without 0.02% MMS. Catalytically “dead” point mutants (C201A and R195G) are as resistant to MMS as the WT allele. <i>(D)</i> Methylation of H3 K36 is not required for resistance to MMS. Five-fold serial dilutions of strains in which histone genes are deleted from the genome but covered by either a plasmid with WT histones (top row) or a plasmid in which H3 carries a K36A point mutation (bottom row) were plated with and without 0.02% MMS. There is no observable difference in survivability between the WT strain and the mutant strain on MMS.</p

Speculative model of the CAR system.

<p><i>(</i><b><i>A</i></b><i>)</i> CAR proteins associate with elongating RNA Polymerase II through interactions with the phosphoCTD. Elongating Pol II (dark blue) is depicted with 3 classes of PCTD associating proteins (shades of pink, brown, and blue, respectively) bound either directly or indirectly to the normally-phosphorylated CTD (dark blue line). We speculate that some CAR proteins form complexes with particular functions; here, for example, we propose that A, B, C, D and E comprise a CAR complex that functions as a damage-responsive module; note how it is coupled to the globular catalytic core of PolII. The elongating catalytic core of Pol II will soon encounter a translocation-blocking DNA lesion (red star on orange DNA). <i>(</i><b><i>B</i></b><i>)</i> CAR proteins respond to damage that blocks elongation. The catalytic core of Pol II has collided with the lesion; <u>translocation is blocked</u>. Changes ensue, signaling that polymerase is blocked and ultimately leading to repair. Possible changes include: 1) alterations in conformation, depicted by shape and surface changes (of core and proteins coupled to it, such as A and B, C, D and E); 2) dissociations and signaling (protein E); 3) changes in activity (not indicated); 4) covalent modifications (beacons on B & D) that signal and/or recruit other components. The combined changes comprise the normal damage response, that leads to repair and normal “damage resistance.” <i>(</i><b><i>C</i></b><i>)</i> An abnormal CAR complex results in aberrant damage response. Here, CAR protein C is truncated, missing its PCTD interacting domain (e.g. SRI domain of Set2). C and E are shown in ghostly white, because in reality they would not properly associate with the elongation complex. Note also that D is no longer coupled to the catalytic core. <i>(</i><b><i>D</i></b><i>)</i> A disrupted CAR system leads to reduced damage resistance. When damage blocks the movement of PolII, changes are induced, but their extent is diminished due to absence of PCTD-binding by mutated protein C. Signaling is reduced (signaling from D and E does not occur). Because only a partial damage response is generated, a reduction in damage resistance is observed. We expect that the specific defects underlying a reduced damage response will differ from case to case, depending on which CAR protein is defective and on the nature of the DNA damage.</p

Interfering with binding of PCAPs to the CTD of elongating RNAPII leads to ionizing radiation sensitivity.

<p><i>(A)</i> Diagrams of CTD-carrying fusion proteins expressed under inducing conditions (in galactose medium) either in the nucleus (nucCTD) or in the cytoplasm (cytoCTD). <i>(B)</i> Percent survival of yeast cells as a function of radiation dose. Liquid cultures of the three strains (three isolates of each in galactose medium) were grown well into stationary phase (G0) and exposed to a single dose of gamma-rays of the indicated intensities. Aliquots were then plated to galactose containing medium and survival was determined by counting colonies after several days at 30°C. Bars indicate standard deviations. <i>(C)</i> Growth of 5-fold serial dilutions expressing nucCTD or cytoCTD after exposure to ionizing radiation.</p