Targeted Covalent Inhibition of Small CTD Phosphatase 1 to Promote the Degradation of the REST Transcription Factor in Human Cells.

The repressor element-1 silencing transcription factor (REST) represses neuronal gene expression, whose dysregulation is implicated in brain tumors and neurological diseases. A high level of REST protein drives the tumor growth in some glioblastoma cells. While transcription factors like REST are challenging targets for small-molecule inhibitors, the inactivation of a regulatory protein, small CTD phosphatase 1 (SCP1), promotes REST degradation and reduces transcriptional activity. This study rationally designed a series of α,ß-unsaturated sulfones to serve as potent and selective covalent inhibitors against SCP1. The compounds inactivate SCP1 via covalent modification of Cys181 located at the active site entrance. Cellular studies showed that the inhibitors inactivate SCP1 in a time- and dose-dependent manner with an EC50 ∼1.5 µM, reducing REST protein levels and activating specific REST-suppressed genes. These compounds represent a promising line of small-molecule inhibitors as a novel lead for glioblastoma whose growth is driven by REST transcription activity.

Advancements in chemical biology targeting the kinases and phosphatases of RNA polymerase II-mediated transcription.

Phosphorylation of RNA polymerase II (RNAP II) coordinates the temporal progression of eukaryotic transcription. The development and application of chemical genetic methods have enhanced our ability to investigate the intricate and intertwined pathways regulated by the kinases and phosphatases targeting RNAP II to ensure transcription accuracy and efficiency. Although identifying small molecules that modulate these enzymes has been challenging due to their highly conserved structures, powerful new chemical biology strategies such as targeted covalent inhibitors and small molecule degraders have significantly improved chemical probe specificity. The recent success in discovering phosphatase holoenzyme activators and inhibitors, which demonstrates the feasibility of selective targeting of individual phosphatase complexes, opens up new avenues into the study of transcription. Herein, we summarize how chemical biology is used to delineate kinases' identities involved in RNAP II regulation and new concepts in inhibitor/activator design implemented for kinases/phosphatases involved in modulating RNAP II-mediated transcription.

Structural determinants for accurate dephosphorylation of RNA polymerase II by its cognate C-terminal domain (CTD) phosphatase during eukaryotic transcription.

The C-terminal domain (CTD) of RNA polymerase II contains a repetitive heptad sequence (YSPTSPS) whose phosphorylation states coordinate eukaryotic transcription by recruiting protein regulators. The precise placement and removal of phosphate groups on specific residues of the CTD are critical for the fidelity and effectiveness of RNA polymerase II-mediated transcription. During transcriptional elongation, phosphoryl-Ser5 (pSer5) is gradually dephosphorylated by CTD phosphatases, whereas Ser2 phosphorylation accumulates. Using MS, X-ray crystallography, protein engineering, and immunoblotting analyses, here we investigated the structure and function of SSU72 homolog, RNA polymerase II CTD phosphatase (Ssu72, from Drosophila melanogaster), an essential CTD phosphatase that dephosphorylates pSer5 at the transition from elongation to termination, to determine the mechanism by which Ssu72 distinguishes the highly similar pSer2 and pSer5 CTDs. We found that Ssu72 dephosphorylates pSer5 effectively but only has low activities toward pSer7 and pSer2 The structural analysis revealed that Ssu72 requires that the proline residue in the substrate's SP motif is in the cis configuration, forming a tight ß-turn for recognition by Ssu72. We also noted that residues flanking the SP motif, such as the bulky Tyr1 next to Ser2, prevent the formation of such configuration and enable Ssu72 to distinguish among the different SP motifs. The phosphorylation of Tyr1 further prohibited Ssu72 binding to pSer2 and thereby prevented untimely Ser2 dephosphorylation. Our results reveal critical roles for Tyr1 in differentiating the phosphorylation states of Ser2/Ser5 of CTD in RNA polymerase II that occur at different stages of transcription.

Phosphatase activity of small C-terminal domain phosphatase 1 (SCP1) controls the stability of the key neuronal regulator RE1-silencing transcription factor (REST).

The RE1-silencing transcription factor (REST) is the major scaffold protein for assembly of neuronal gene silencing complexes that suppress gene transcription through regulating the surrounding chromatin structure. REST represses neuronal gene expression in stem cells and non-neuronal cells, but it is minimally expressed in neuronal cells to ensure proper neuronal development. Dysregulation of REST function has been implicated in several cancers and neurological diseases. Modulating REST gene silencing is challenging because cellular and developmental differences can affect its activity. We therefore considered the possibility of modulating REST activity through its regulatory proteins. The human small C-terminal domain phosphatase 1 (SCP1) regulates the phosphorylation state of REST at sites that function as REST degradation checkpoints. Using kinetic analysis and direct visualization with X-ray crystallography, we show that SCP1 dephosphorylates two degron phosphosites of REST with a clear preference for phosphoserine 861 (pSer-861). Furthermore, we show that SCP1 stabilizes REST protein levels, which sustains REST's gene silencing function in HEK293 cells. In summary, our findings strongly suggest that REST is a bona fide substrate for SCP1 in vivo and that SCP1 phosphatase activity protects REST against degradation. These observations indicate that targeting REST via its regulatory protein SCP1 can modulate its activity and alter signaling in this essential developmental pathway.

Mapping the Phosphorylation Pattern of Drosophila melanogaster RNA Polymerase II Carboxyl-Terminal Domain Using Ultraviolet Photodissociation Mass Spectrometry.

Phosphorylation of the C-terminal domain of RNA polymerase II (CTD) plays an essential role in eukaryotic transcription by recruiting transcriptional regulatory factors to the active polymerase. However, the scarcity of basic residues and repetitive nature of the CTD sequence impose a huge challenge for site-specific characterization of phosphorylation, hindering our understanding of this crucial biological process. Herein, we apply LC-UVPD-MS methods to analyze post-translational modification along native sequence CTDs. Application of our method to the Drosophila melanogaster CTD reveals the phosphorylation pattern of this model organism for the first time. The divergent nature of fly CTD allows us to derive rules defining how flanking residues affect phosphorylation choice by CTD kinases. Our data support the use of LC-UVPD-MS to decipher the CTD code and determine rules that program its function.

Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase.

Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.