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Principal Investigator: ROBERT P FISHER
Organization: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
Fiscal Year: 2024
Award: $507,000
Funding agency: National Institute of General Medical Sciences
Project Summary
RNA polymerase II (RNAPII), which catalyzes mRNA synthesis, is co-regulated by cyclin-dependent kinases
(CDKs) and phosphatases to control transcription cycle progression and ensure coupling of elongation and
processing. Derangements of the RNAPII cycle underlie cancer and other diseases; CDKs and phosphatases
are potential drug targets, but how they collaborate to regulate gene expression is largely unknown. We seek
to determine how specific phosphorylations are placed and removed in precise order to execute transitions
between phases of transcription. In the past five years we uncovered two CDK-phosphatase switches that
regulate RNAPII elongation. Our goals for the next five years are to understand how these circuits work to
coordinate the transcription cycle, and how they might be disrupted with small molecules.
Dissecting CDK-PPP switches: We identified two members of the phosphoprotein phosphatase (PPP) family
as targets of inhibitory phosphorylation by Cdk9 (P-TEFb). PP4 and PP1 dephosphorylate the Cdk9 substrate
Spt5 to regulate the onset and end of rapid elongation, respectively. To define roles of Cdk9-PPP crosstalk in
transcription, we will introduce mutations that lock PP4 or PP1 in constitutively active (unphosphorylated) or
inactive (phosphorylated) states and analyze genome-wide effects on RNAPII dynamics and gene expression.
Unraveling control of RNAPII elongation by PPP family phosphatases: PP4 and the related PP2A,
another phosphatase recruited to transcription complexes, dephosphorylate Spt5 with similar site specificity,
and may cooperate to regulate RNAPII release from the promoter-proximal pause. We plan integrated genomic
analyses to ask whether PP4 and PP2A work independently, in combination, or in response to different signals.
Understanding how phosphorylation events are ordered in the RNAPII cycle: By analogy with the cell
division cycle—also regulated by CDKs—the order of phosphorylations during transcription is likely determined
by substrate specificities of CDKs and phosphatases, and by structural features of the substrates themselves.
We will test this hypothesis for Cdk9, which has scores of substrates and is active throughout elongation.
Functional dissection of a conserved elongation regulator: Spt5, large subunit of the conserved elongation
factor DSIF, is the linchpin of elongation control by Cdk9-PPP switches; phosphorylations placed by Cdk9 at
different sites in Spt5 are removed at different times by PP4 or PP1 and are likely to perform different functions
in the transcription cycle. We will take a genome editing approach in human cells to dissect the functions of
distinct Spt5 phosphorylations, and to elucidate their differential regulation by CDKs and phosphatases.
Illuminating a link between transcription and cell cycle control: One CDK is a core component of both
cell-division and transcriptional machineries: the TFIIH-associated Cdk7. As human cells re-enter the division
cycle from quiescence, chromatin-bound Cdk7 is activated, potentially enhancing its function in G1-specific
gene transcription. We will test this model and identify pathways that lead to Cdk7 activation at cell cycle entry.
Terms: <BTF2 transcription factor><Binding><Cancer Treatment><Cancers><Cell Body><Cell Communication and Signaling><Cell Cycle><Cell Cycle Control><Cell Cycle Regulation><Cell Division Cycle><Cell Signaling><Cell division><Cells><Chromatin><Code><Coding System><Collaborations><Complex><Coupling><Cyclin-Dependent Kinases><Cyclin-Dependent Protein Kinases><DNA-Dependent RNA Polymerase II><Dephosphorylation><Disease><Disorder><Dissection><Drug Targeting><Elongation Factor><Ensure><Event><Family><Gene Action Regulation><Gene Expression><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Gene Transcription><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Genomics><Goals><Growth and Development><Growth and Development function><Human><Intracellular Communication and Signaling><Link><Malignant Cell><Malignant Neoplasm Therapy><Malignant Neoplasm Treatment><Malignant Neoplasms><Malignant Tumor><Messenger RNA><Modeling><Modern Man><Molecular Interaction><Mutation><PP2A><PP2A Subunit B Prime><Pathway interactions><Phase><Phosphatases><Phosphohydrolases><Phosphomonoesterases><Phosphoprotein Phosphatase><Phosphoprotein Phosphatase-2C><Phosphoprotein Phosphohydrolase><Phosphoric Monoester Hydrolases><Phosphorylation><Phosphotyrosyl Phosphatase Activator><Protein Dephosphorylation><Protein Phosphatase 2A Regulatory Subunit B Prime><Protein Phosphatase 2A Regulatory Subunit PR53><Protein Phosphatase C><Protein Phosphatase Gene><Protein Phosphatase-1><Protein Phosphatase-2A><Protein Phosphorylation><Protein phosphatase><RNA Expression><RNA Polymerase B><RNA Polymerase II><Regulation><Role><Signal Transduction><Signal Transduction Systems><Signaling><Site><Specificity><Substrate Specificity><TFIIH><Testing><Therapeutic><Transcription><Transcription Regulation><Transcriptional Control><Transcriptional Regulation><Work><anti-cancer therapy><basic transcription factor 2><biological signal transduction><cancer cell><cancer therapy><cancer-directed therapy><cdk Proteins><genome editing><genome mutation><genome scale><genome-wide><genomewide><genomic editing><mRNA><malignancy><member><neoplasm/cancer><pathway><phosphatase-1 kinase><phosphoprotein phosphatase kinase><promoter><promotor><protein expression><recruit><response><small molecule><social role><transcription factor IIH><transcription factor TFIIH>