G. Sebastiaan Winkler

Elongator is a histone H3 and H4 acetyltransferase important for normal histone acetylation levels in vivo

The elongating, hyperphosphorylated form of RNA polymerase II is associated with the Elongator complex, which has the histone acetyltransferase (HAT) Elp3 as a subunit. Here we show that, in contrast to the isolated Elp3 subunit, the activity of intact Elongator complex is directed specifically toward the amino-terminal tails of histone H3 and H4, and that Elongator can acetylate both core histones and nucleosomal substrates. The predominant acetylation sites are lysine-14 of histone H3 and lysine-8 of histone H4. The three smallest Elongator subunits—Elp4, Elp5, and Elp6—are required for HAT activity, and Elongator binds to both naked and nucleosomal DNA. By using chromatin immunoprecipitation, we show that the levels of multiply acetylated histone H3 and H4 in chromatin are decreased in vivo in yeast cells lacking ELP3.

The mammalian anti-proliferative BTG/Tob protein family.

The mammalian BTG/Tob family comprises six proteins (BTG1, BTG2/PC3/Tis21, BTG3/ANA, BTG4/PC3B, Tob1/Tob and Tob2), which regulate cell cycle progression in a variety of cell types. They are characterised by the conserved N‐terminal domain spanning 104–106 amino acids. Recent biochemical and structural data indicate that the conserved BTG domain is a protein–protein interaction module, which is capable of binding to DNA‐binding transcription factors as well as the paralogues CNOT7 (human Caf1/Caf1a) and CNOT8 (human Pop2/Calif/Caf1b), two deadenylase subunits of the Ccr4‐Not complex. Consistent with this finding, several members of the BTG/Tob family are shown to be implicated in transcription in the nucleus and cytoplasmic mRNA deadenylation and turnover. The C‐terminal regions are less conserved and appear to mediate protein–protein interactions that are unique to each family member. The human and mouse BTG/Tob proteins will be the focus of this review and structural aspects of BTG/Tob interactions with components of the Ccr4‐Not complex, and the role of the BTG/Tob proteins in the regulation of gene expression, tumourigenesis and cancer will be discussed. J. Cell. Physiol. 222:66–72, 2010.

Menin Links Estrogen Receptor Activation to Histone H3K4 Trimethylation

The product of the multiple endocrine neoplasia type 1 (MEN1) tumor suppressor gene, menin, is an integral component of MLL1/MLL2 histone methyltransferase complexes specific for Lys4 of histone H3 (H3K4). We show that menin is a transcriptional coactivator of the nuclear receptors for estrogen and vitamin D. Activation of the endogenous estrogen-responsive TFF1 (pS2) gene results in promoter recruitment of menin and in elevated trimethylation of H3K4. Knockdown of menin reduces both activated TFF1 (pS2) transcription and H3K4 trimethylation. In addition, menin can directly interact with the estrogen receptor-alpha (ERalpha) in a hormone-dependent manner. The majority of disease-related MEN1 mutations prevent menin-ERalpha interaction. Importantly, ERalpha-interacting mutants are also defective in coactivator function. Our results indicate that menin is a critical link between recruitment of histone methyltransferase complexes and nuclear receptor-mediated transcription.

TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair

TFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and XPD, are associated with three inherited syndromes as follows: xeroderma pigmentosum with or without Cockayne syndrome and trichothiodystrophy. By using epitope-tagged XPD we purified mammalian TFIIH carrying a wild type or an active-site mutant XPD subunit. Contrary to XPB, XPD helicase activity was dispensable for in vitro transcription, catalytic formation of trinucleotide transcripts, and promoter opening. Moreover, in contrast to XPB, microinjection of mutant XPD cDNA did not interfere with in vivo transcription. These data show directly that XPD activity is not required for transcription. However, during DNA repair, neither 5′ nor 3′ incisions in defined positions around a DNA adduct were detected in the presence of TFIIH containing inactive XPD, although substantial damage-dependent DNA synthesis was induced by the presence of mutant XPD both in cells and cell extracts. The aberrant damage-dependent DNA synthesis caused by the mutant XPD does not lead to effective repair, consistent with the discrepancy between repair synthesis and survival in cells from a number of XP-D patients.

Elongator Interactions with Nascent mRNA Revealed by RNA Immunoprecipitation

The histone acetyltransferase Elongator was originally isolated as a component of the elongating form of RNA polymerase II (RNAPII) and a plethora of data has since supported a role for the factor in transcription. However, recent data has suggested that it is predominantly cytoplasmic and does not associate with the DNA of transcribed genes in vivo. Here, we report that Elongator binds to RNA both in vitro and in vivo. Using a modified chromatin immunoprecipitation procedure, RNA immunoprecipitation (RIP), we show that Elongator is indeed present at several actively transcribed genes and that it associates with the nascent RNA emanating from elongating RNAPII along the entire coding region of a gene. These results strongly support a role for Elongator in transcript elongation.

Isolation and mass spectrometry of transcription factor complexes.

Protocols are described that enable the isolation of novel proteins associated with a known protein and the subsequent identification of these proteins by mass spectrometry. We review the basics of nanosample handling and of two complementary approaches to mass analysis, and provide protocols for the entire process. The protein isolation procedure is rapid and based on two high-affinity chromatography steps. The method does not require previous knowledge of complex composition or activity and permits subsequent biochemical characterization of the isolated factor. As an example, we provide the procedures used to isolate and analyze yeast Elongator, a histone acetyltransferase complex important for transcript elongation, which led to the identification of three novel subunits.

The Ccr4–Not Deadenylase Subunits CNOT7 and CNOT8 Have Overlapping Roles and Modulate Cell Proliferation

Accurate gene expression requires the precise control of mRNA levels, which are determined by the relative rates of nuclear (pre-)mRNA synthesis and processing, and cytoplasmic mRNA turnover. A key step in mRNA degradation is the removal of the poly(A) tail, which involves several deadenylases including components of the Ccr4-Not complex. Here, we focused on the role of the human paralogues CNOT7 (hCaf1/Caf1a) and CNOT8 (hPop2/Caf1b/Calif), which possess deadenylase activity mediated by DEDD nuclease domains. We show that efficient proliferation requires both subunits, although combined knockdown of CNOT7 and CNOT8 further reduces cell proliferation indicating partial redundancy between these proteins. Interestingly, the function of CNOT7 in cell proliferation partly depends on its catalytic activity. On the other hand, the interaction between CNOT7 and BTG2, a member of the antiproliferative BTG/Tob family involved in transcription and mRNA decay appears less important for proliferation of MCF7 cells, suggesting that CNOT7 does not function solely in conjunction with BTG2. Further analysis of gene expression profiles of CNOT7 and/or CNOT8 knockdown cells underscores the partial redundancy between these subunits and suggests that regulation of several genes, including repression of the antiproliferative genes MSMB and PMP22, by the Ccr4-Not complex contributes to cell proliferation.

Human Ccr4-Not complex is a ligand-dependent repressor of nuclear receptor-mediated transcription

The Ccr4‐Not complex is a highly conserved regulator of mRNA metabolism. The transcription regulatory function of this complex in higher eukaryotes, however, is largely unexplored. Here we report that CNOT1, the large human subunit, represses the ligand‐dependent transcriptional activation function of oestrogen receptor (ER) α. Promoter recruitment assays indicate that CNOT1 contains an intrinsic ability to mediate transcriptional repression. Furthermore, CNOT1 can interact with the ligand‐binding domain of ERα in a hormone‐dependent fashion and is recruited with other Ccr4‐Not subunits to endogenous oestrogen‐regulated promoters dependent on the presence of ligand. In addition, siRNA‐mediated depletion of endogenous CNOT1 or other Ccr4‐Not subunits in breast cancer cells results in deregulation of ERα target genes. Finally, CNOT1 interacts in a ligand‐dependent manner with RXR and represses transcription mediated by several RXR heterodimers. These findings define a function for the human Ccr4‐Not complex as a transcriptional repressor of nuclear receptor signalling that is relevant for the understanding of molecular pathways involved in cancer.

The Ccr4a (CNOT6) and Ccr4b (CNOT6L) deadenylase subunits of the human Ccr4–Not complex contribute to the prevention of cell death and senescence

The human Ccr4-Not complex has two types of deadenylase subunits that shorten the polyadenylate tail of cytoplasmic mRNA. The authors present evidence for novel roles of the highly related Ccr4a/Ccr4b deadenylases in preventing cell death and senescence and show that they have distinct roles as compared with the Caf1a/Caf1b deadenylases.

DNA damage and replication stress induced transcription of RNR genes is dependent on the Ccr4–Not complex

Genetic experiments have indicated a role for the Ccr4–Not complex in the response to hydroxyurea (HU) induced replication stress and ionizing radiation in yeast. This response includes transcriptional induction of the four genes constituting the ribonucleotide reductase (RNR) enzymatic complex, RNR1-4 and degradation of its inhibitor, Sml1p. The Ccr4–Not complex has originally been described as a negative regulator of RNA polymerase II (pol II) transcription, but it has also been implicated in mRNA turnover and protein ubiquitination. We investigated the mechanism of the HU sensitivity conferred by mutation of CCR4-NOT genes. We found that the ubiquitin protein ligase activity of Not4p does not play a role in HU induced Sml1p degradation. We show, however, that the HU sensitivity of ccr4-not mutant strains correlated very well with a defect in accumulation of RNR2, RNR3 and RNR4 mRNA after HU or methyl-methane sulfonate (MMS) treatment. Chromatin immunoprecipitation (ChIP) experiments show that TBP, pol II and Set1p recruitment to the activated RNR3 locus is defective in cells lacking NOT4. Moreover, RNR3-promoter activity is not induced by HU in these cells. Our experiments show that induction of RNR gene transcription is defective in ccr4-not mutant strains, providing an explanation for their sensitivity to HU.