Hyperphosphorylated C-terminal Repeat Domain-associating Proteins in the Nuclear Proteome Link Transcription to DNA/Chromatin Modification and RNA Processing

Duke University, Durham, North Carolina, United States
Molecular &amp Cellular Proteomics (Impact Factor: 6.56). 09/2002; 1(8):598-610. DOI: 10.1074/mcp.M200029-MCP200
Source: PubMed


Using an interaction blot approach to search in the human nuclear proteome, we identified eight novel proteins that bind the hyperphosphorylated C-terminal repeat domain (phosphoCTD) of RNA polymerase II. Unexpectedly, five of the new phosphoCTD-associating proteins (PCAPs) represent either enzymes that act on DNA and chromatin (topoisomerase I, DNA (cytosine-5) methyltransferase 1, poly(ADP-ribose) polymerase-1) or proteins known to bind DNA (heterogeneous nuclear ribonucleoprotein (hnRNP) U/SAF-A, hnRNP D). The other three PCAPs represent factors involved in pre-mRNA metabolism as anticipated (CA150, NSAP1/hnRNP Q, hnRNP R) (note that hnRNP U/SAF-A and hnRNP D are also implicated in pre-mRNA metabolism). Identifying as PCAPs proteins involved in diverse DNA transactions suggests that the range of phosphoCTD functions extends far beyond just transcription and RNA processing. In view of the activities possessed by the DNA-directed PCAPs, it is likely that the phosphoCTD plays important roles in genome integrity, epigenetic regulation, and potentially nuclear structure. We present a model in which the phosphoCTD association of the PCAPs poises them to act either on the nascent transcript or on the DNA/chromatin template. We propose that the phosphoCTD of elongating RNA polymerase II is a major organizer of nuclear functions.

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    • "In addition, proper phosphorylation of the CTD by CTDK-I (CTD kinase I) is required for normal levels of resistance to several chemical and physical damaging agents in yeast (see Table S1). Moreover, a number of phosphoCTD-associating proteins (PCAPs) are already known to be required for normal resistance to DNA damaging agents or are otherwise involved in DNA repair/genome stability; these include yeast PCAPs Ess1, Hrr25, Chl1, Pms1, Rtt103, Sen1 and TopoI [8], [9], [18]–[21], and metazoan PCAPs PARP1, TopoI, RecQ5 and ASF/SF2 [22]–[27]. Finally, deletions of genes for any one of the three CTDK-I subunits (Ctk1, Ctk2 or Ctk3) are synthetically lethal with individual deletions of a large number of “DNA integrity” genes (see Table S2). These interactions imply functional relationships between CTDK-I and numerous repair proteins, including those involved in homologous recombination (HR)-mediated repair. "
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    ABSTRACT: 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 CTK1, 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 CTD-Associated DNA damage Response (CAR) system, organized around the phosphoCTD of elongating RNAPII and comprising a subset of phosphoCTD-associating proteins.
    PLoS ONE 04/2013; 8(4):e60909. DOI:10.1371/journal.pone.0060909 · 3.23 Impact Factor
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    • "The recruitment of Spt6 follows 10 s after Pol II, which is consistent with the known role of Spt6 as a chromatin remodeler and the time for Pol II to reach the first nucleosome. Then Topo I, which interacts with the phosphorylated CTD of Pol II (Carty and Greenleaf, 2002), is recruited 20 s after Pol II and P-TEFb. "
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    ABSTRACT: Chromatin immunoprecipitation (ChIP) studies provide snapshots of factors on chromatin in cell populations. Here, we use live-cell imaging to examine at high temporal resolution the recruitment and dynamics of transcription factors to the inducible Hsp70 loci in individual Drosophila salivary gland nuclei. Recruitment of the master regulator, HSF, is first detected within 20 s of gene activation; the timing of its recruitment resolves from RNA polymerase II and P-TEFb, and these factors resolve from Spt6 and Topo I. Remarkably, the recruitment of each factor is highly synchronous between different cells. In addition, fluorescence recovery after photobleaching (FRAP) analyses show that the entry and exit of multiple factors are progressively constrained upon gene activation, suggesting the gradual formation of a transcription compartment. Furthermore, we demonstrate that poly(ADP-ribose) (PAR) polymerase activity is required to maintain the transcription compartment. We propose that PAR polymers locally retain factors in a transcription compartment.
    Molecular cell 12/2010; 40(6):965-75. DOI:10.1016/j.molcel.2010.11.022 · 14.02 Impact Factor
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    • "RNA polymerase II is a central component of a complex apparatus responsible for controlling the basic biochemical steps required for mRNA production, including transcription initiation, capping, elongation, splicing, and polyadenylation (Howe, 2002;Zorio and Bentley 2004;Proudfoot, Furger et al. 2002). These diverse processes are integrated by the tail of the molecule, referred to as the C-terminal domain (CTD) of the protein (Carty and Greenleaf 2002). The CTD, essential for gene expression in animals and fungi, is comprised of a tandem array of heptapeptide repeats, featuring a signature amino acid sequence: Y 1 -S 2 -P 3 -T 4 -S 5 -P 6 -S 7 (Corden, 1990;Allison, Moyle et al. 1985). "
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    ABSTRACT: The tail of the enzyme RNA polymerase II is responsible for integrating the diverse events of gene expression in eukaryotes and is indispensable for life in yeast, fruit flies, and mice. The tail features a C-terminal domain (CTD), which is comprised of tandemly repeated Y(1)-S(2)-P(3)-T(4)-S(5)-P(6)-S(7) amino acid heptads that are highly conserved across evolutionary lineages, with all mammalian polymerases featuring 52 identical heptad repeats. However, the composition and function of protozoan CTDs remain less well understood. We find that malaria parasites (genus Plasmodium) display an unprecedented plasticity within the length and composition of their CTDs. The CTD in malaria parasites which infect human and nonhuman primates has expanded compared to closely related species that infect rodents or birds. In addition, this variability extends to different isolates within a single species, such as isolates of the human malaria parasite, Plasmodium falciparum. Our results indicate that expanded CTD heptads in malaria parasites correlates with parasitism of primates and provide the first demonstration of polymorphism of the RNA polymerase II CTD within a single species. The expanded set of CTD heptads feature lysine in the seventh position (Y(1)-S(2)-P(3)-T(4)-S(5)-P(6)-K(7)), a sequence only seen otherwise in the distal portion of mammalian polymerases. These observations raise new questions for the radiation of malaria parasites into diverse hosts and for the molecular evolution of RNA polymerase II.
    Journal of Molecular Evolution 06/2009; 68(6):706-14. DOI:10.1007/s00239-009-9245-2 · 1.68 Impact Factor
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