RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Differential phosphorylation of serine-2 and serine-5 at the 5' and 3' regions of genes appears to coordinate the localization of transcription and RNA processing factors to the elongating polymerase complex. Using monoclonal antibodies, we reveal serine-7 phosphorylation on transcribed genes. This position does not appear to be phosphorylated in CTDs of less than 20 consensus repeats. The position of repeats where serine-7 is substituted influenced the appearance of distinct phosphorylated forms, suggesting functional differences between CTD regions. Our results indicate that restriction of serine-7 epitopes to the Linker-proximal region limits CTD phosphorylation patterns and is a requirement for optimal gene expression.

Recent work has shown that RNA polymerase (Pol) II can be recruited to and transcribe distal regulatory regions. Here we analyzed transcription initiation and elongation through genome-wide localization of Pol II, general transcription factors (GTFs) and active chromatin in developing T cells. We show that Pol II and GTFs are recruited to known T cell-specific enhancers. We extend this observation to many new putative enhancers, a majority of which can be transcribed with or without polyadenylation. Importantly, we also identify genomic features called transcriptional initiation platforms (TIPs) that are characterized by large areas of Pol II and GTF recruitment at promoters, intergenic and intragenic regions. TIPs show variable widths (0.4-10 kb) and correlate with high CpG content and increased tissue specificity at promoters. Finally, we also report differential recruitment of TFIID and other GTFs at promoters and enhancers. Overall, we propose that TIPs represent important new regulatory hallmarks of the genome.

The RING ®nger protein CNOT4 is a component of the CCR4±NOT complex. This complex is implicated in repression of RNA polymerase II transcription. Here we demonstrate that CNOT4 functions as a ubiquitin±protein ligase (E3). We show that the unique C 4 C 4 RING domain of CNOT4 interacts with a subset of ubiquitin-conjugating enzymes (E2s). Using NMR spectroscopy, we detail the interaction of CNOT4 with UbcH5B and characterize RING residues that are critical for this interaction. CNOT4 acts as a potent E3 ligase in vitro. Mutations that destabilize the E2±E3 interface abolish this activity. Based on these results, we present a model of how E3 ligase function within the CCR4±NOT complex relates to transcriptional regulation.

The yeast CCR4-NOT protein complex is a global regulator of RNA polymerase II transcription. It is comprised of yeast NOT1 to NOT5, yeast CCR4 and additional proteins like yeast CAF1. Here we report the isolation of cDNAs encoding human NOT2, NOT3, NOT4 and a CAF1-like factor, CALIF. Analysis of their mRNA levels in different human tissues reveals a common ubiquitous expression pattern. A multitude of two-hybrid interactions among the human cDNAs suggest that their encoded proteins also form a complex in mammalian cells. Functional conservation of these proteins throughout evolution is supported by the observation that the isolated human NOT3 and NOT4 cDNAs can partially com-plement corresponding not mutations in yeast. Interestingly, human CALIF is highly homologous to, although clearly different from, a recently described human CAF1 protein. Conserved interactions of this factor with both NOT and CCR4 proteins and co-immunoprecipitation experiments suggest that CALIF is a bona fide component of the human CCR4-NOT complex.

BACKGROUND AND PURPOSEThe cyclin-dependent kinase CDK9 is an important therapeutic target but currently available inhibitors exhibit low specificity and/or narrow therapeutic windows. Here we have used a new highly specific CDK9 inhibitor, LDC000067 to interrogate gene control mechanisms mediated by CDK9. EXPERIMENTAL APPROACHThe selectivity of LDC000067 was established in functional kinase assays. Functions of CDK9 in gene expression were assessed with in vitro transcription experiments, single gene analyses and genome-wide expression profiling. Cultures of mouse embryonic stem cells, HeLa cells, several cancer cell lines, along with cells from patients with acute myelogenous leukaemia were also used to investigate cellular responses to LDC000067. KEY RESULTSThe selectivity of LDC000067 for CDK9 over other CDKs exceeded that of the known inhibitors flavopiridol and DRB. LDC000067 inhibited in vitro transcription in an ATP-competitive and dose-dependent manner. Gene expression profiling of cells treated with LDC000067 demonstrated a selective reduction of short-lived mRNAs, including important regulators of proliferation and apoptosis. Analysis of de novo RNA synthesis suggested a wide ranging positive role of CDK9. At the molecular and cellular level, LDC000067 reproduced effects characteristic of CDK9 inhibition such as enhanced pausing of RNA polymerase II on genes and, most importantly, induction of apoptosis in cancer cells. CONCLUSIONS AND IMPLICATIONSOur study provides a framework for the mechanistic understanding of cellular responses to CDK9 inhibition. LDC000067 represents a promising lead for the development of clinically useful, highly specific CDK9 inhibitors. AbbreviationsLDC067, LDC000067, 3-((6-(2-methoxyphenyl)pyrimidin-4-yl)amino)phenyl) methane sulfonamide; AML, acute myelogenous leukaemia; CDK, cyclin-dependent kinase; ChIP, chromatin immunoprecipitation; CTD, carboxyterminal domain; DRB, 5,6-dichloro-1-β-D-ribofuranosyl-benzimidazole; DSIF, DRB sensitivity-inducing factor; FRET, fluorescence resonance energy transfer; mESCs, mouse embryonic stem cells; NELF, negative elongation factor; P-TEFb, positive transcription elongation factor b; RNAPII, RNA polymerase II; RT-qPCR, reverse transcription-quantitative real-time PCR; Ser2/5/7-P, CTD phospho-Ser 2/5/7

b Cyclin-dependent kinase 7 (CDK7) activates cell cycle CDKs and is a member of the general transcription factor TFIIH. Although there is substantial evidence for an active role of CDK7 in mRNA synthesis and associated processes, the degree of its influence on global and gene-specific transcription in mammalian species is unclear. In the current study, we utilize two novel inhibitors with high specificity for CDK7 to demonstrate a restricted but robust impact of CDK7 on gene transcription in vivo and in in vitro-reconstituted reactions. We distinguish between relative low-and high-dose responses and relate them to distinct molecular mechanisms and altered physiological responses. Low inhibitor doses cause rapid clearance of paused RNA polymerase II (RNAPII) molecules and sufficed to cause genome-wide alterations in gene expression, delays in cell cycle progression at both the G 1 /S and G 2 /M checkpoints, and diminished survival of human tumor cells. Higher doses and prolonged inhibition led to strong reductions in RNAPII carboxyl-terminal domain (CTD) phosphorylation, eventual activation of the p53 program, and increased cell death. Together, our data reason for a quantitative contribution of CDK7 to mRNA synthesis, which is critical for cellular homeostasis. C yclin-dependent kinases (CDKs) form the enzymatic components of a group of heterodimeric serine/threonine kinases that have important roles in multiple cellular processes (1). CDK7/KIN28 was originally identified as a critical regulator of mRNA transcription in Saccharomyces cerevisiae (2-5). In vertebrates CDK7 has a dual function, influencing cell cycle progression and RNA polymerase II (RNAPII) transcription (6). Specifically, CDK7 forms the CDK-activating kinase (CAK) with two other TFIIH subunits, cyclin H and MAT1. The CAK activates downstream cell cycle CDKs, including cdc-2/CDK1, CDK2, CDK4, and CDK6, by phosphorylating key threonine residues in a process known as T-loop activation (7,8). In transcription, as RNAPII begins to lose contact with many of the general transcription factors (GTFs) during promoter escape, CDK7, functioning as part of the TFIIH complex, phosphorylates RNAPII and allows the elongation complex to move downstream away from the transcription start site (TSS) (reviewed in reference 9). Specifically, CDK7 directly targets the carboxyl-terminal domain (CTD) of the Rpb1 subunit of RNAPII, which is comprised of 52 heptad repeats (Y 1 S 2 P 3 T 4 S 5 P 6 S 7 ) in humans. While the serine 2 (Ser2), serine 5 (Ser5), and serine 7 (Ser7) residues are all subject to phosphorylation, CDK7 preferentially targets Ser5 and Ser7 (10-17). Phosphorylation patterning of the CTD is important as it influences the association of numerous nuclear factors with RNAPII (18,19), as was recently demonstrated in yeast, where KIN28-driven phosphorylation of Ser5 residues was shown to trigger dissociation of the coactivator Mediator (20). In mammals, the exact mechanisms linking CTD phosphorylation (CTD-P) with transcription are yet to be fully elucidate...

The NOT4 protein is a component of the CCR4⅐NOT complex, a global regulator of RNA polymerase II transcription. Human NOT4 (hNOT4) contains a RING finger motif of the C 4 C 4 type. We expressed and purified the N-terminal region of hNOT4 (residues 1-78) encompassing the RING finger motif and determined the solution structure by heteronuclear NMR. NMR experiments using a 113 Cd-substituted hNOT4 RING finger showed that two metal ions are bound through cysteine residues in a cross-brace manner. The three-dimensional structure of the hNOT4 RING finger was refined with root mean square deviation values of 0.58 ؎ 0.13 Å for the backbone atoms and 1.08 ؎ 0.12 Å for heavy atoms. The hNOT4 RING finger consists of an ␣-helix and three long loops that are stabilized by zinc coordination. The overall folding of the hNOT4 RING finger is similar to that of the C 3 HC 4 RING fingers. The relative orientation of the two zinc-chelating loops and the ␣-helix is well conserved. However, for the other regions, the secondary structural elements are distinct.The CCR4⅐NOT complex was first detected in Saccharomyces cerevisiae as a global transcription regulator, affecting transcription of multiple functionally unrelated genes positively as well as negatively (1). The complex consists of CCR4 (carbon catabolite repressor 4), CAF1 (CCR4-associated factor 1, also known as POP2), the five NOT proteins (NOT1-5), and several unidentified proteins (1). The yeast NOT genes have been identified in a screen for elevated HIS3 expression (2-4). The HIS3 gene contains two core promoters, T C , a TATA-less element, and T R , a canonical TATA sequence (5, 6). Mutations in NOT genes selectively elevate transcription from T C (2-4). Besides repressing genes involved in histidine biosynthesis (HIS3 and HIS4), NOT proteins also affect transcription of genes involved in pheromone response (STE4), nuclear fusion (BIK1), and RNA polymerase II transcription (TBP) (2, 3). The CCR4 gene product regulates expression of ADH2 and other genes involved in nonfermentative growth, cell wall integrity, and ion sensitivity (7-9). CCR4 exists in a complex with other proteins (10), and two-hybrid screening with CCR4 identified CAF1 (11, 12) and DBF2 (a cell cycle-regulated kinase) (9, 13) as binding partners. Recently, it was found that CCR4 and CAF1 reside with the NOT proteins in a 1.2-MDa complex (1). Besides physical interactions between CCR4, CAF1, and NOT proteins, there is also a functional association. Mutations in the NOT, CCR4, and CAF1 genes lead to similar, but not identical, phenotypes (1,14). Interestingly, mutations in NOT1, NOT3, NOT5, and CAF1 genes suppressed a mutation in SRB4, which is an essential component of the RNA polymerase II holoenzyme and required for the expression of most protein-coding genes. This suggests that the yeast CCR4⅐NOT complex has a very general role in RNA polymerase II transcription (15).Recently, the human counterpart of the yeast CCR4⅐NOT complex has been identified (16). cDNAs for four subunits, hNOT2, 1 hNOT3, hNOT4, and...

The proto-oncogene c-myc is transcribed from a dual promoter P1/P2, with transcription initiation sites 160 base pairs apart. Here we have studied the transcriptional activation of both promoters on chromatin templates. c-myc chromatin was reconstituted on stably transfected, episomal, Epstein-Barr virus-derived vectors in a B cell line. Episomal P1 and P2 promoters showed only basal activity but were strongly inducible by histone deacetylase inhibitors. The effect of promoter mutations on c-myc activity, chromatin structure, and E2F binding was studied. The ME1a1 binding site between P1 and P2 was required for the maintenance of an open chromatin configuration of the dual c-myc promoters. Mutation of this site strongly reduced the sensitivity of the core promoter region of P1/P2 to micrococcal nuclease and prevented binding of polymerase II (pol II) at the P2 promoter. In contrast, mutation of the P2 TATA box also abolished binding of pol II at the P2 promoter but did not affect the chromatin structure of the P1/P2 core promoter region. The E2F binding site adjacent to ME1a1 is required for repression of the P2 promoter but not the P1 promoter, likely by recruitment of histone deacetylase activity. Chromatin precipitation experiments with E2F-specific antibodies revealed binding of E2F-1, E2F-2, and E2F-4 to the E2F site of the c-myc promoter in vivo if the E2F site was intact. Taken together, the analyses support a model with a functional hierarchy for regulatory elements in the c-myc promoter region; binding of proteins to the ME1a1 site provides a nucleosome-free region of chromatin near the P2 start site, binding of E2F results in transcriptional repression without affecting polymerase recruitment, and the TATA box is required for polymerase recruitment.The nucleosomal structure of promoter regions constitutes an essential regulatory mechanism of eukaryotic gene repression. Gene activation is accompanied by perturbations or alterations of the nucleosomal structure like remodeling and acetylation of chromatin (1, 2). A first critical step for activating a gene locus seems to be liberating the promoter region from histone-mediated repression (1, 2). The transcription complex seems not to be recruited to the promoter until positioned nucleosomes have been disrupted by the concerted action of invading chromatin-remodeling and trans-acting factors (1, 2). A large number of chromatin remodeling activities have been isolated and characterized biochemically. The properties of remodeled nucleosomes include increased accessibility of DNA to DNA-binding proteins throughout the nucleosome (3-5), rotational phasing of DNA on the histone octamer (6), reduction of the total length of DNA per nucleosome (7), octamer susceptibility to displacement in trans (8), nucleosome movement without disruption or trans-displacement of histone octamer (9), and other effects.Chromatin remodeling activities transfer promoters into a configuration that is accessible for sequence-specific binding of transcription factors and the recruitmen...