RESUMO
Organisms that count X-chromosome number to determine sex utilize dosage compensation mechanisms to balance X-gene expression between sexes. Typically, a regulatory complex is recruited to X chromosomes of one sex to modulate gene expression. A major challenge is to determine the mechanisms that target regulatory complexes specifically to X. Here, we identify critical X-sequence motifs in Caenorhabditis elegans that act synergistically in hermaphrodites to direct X-specific recruitment of the dosage compensation complex (DCC), a condensin complex. We find two DNA motifs that collaborate with a previously defined 12-bp motif called MEX (motif enriched on X) to mediate binding: MEX II, a 26-bp X-enriched motif and Motif C, a 9-bp motif that lacks X enrichment. Inserting both MEX and MEX II into a new location on X creates a DCC binding site equivalent to an endogenous recruitment site, but inserting only MEX or MEX II alone does not. Moreover, mutating MEX, MEX II, or Motif C in endogenous recruitment sites with multiple different motifs dramatically reduces DCC binding in vivo to nearly the same extent as mutating all motifs. Changing the orientation or spacing of motifs also reduces DCC binding. Hence, synergy in DCC binding via combinatorial clustering of motifs triggers DCC assembly specifically on X chromosomes. Using an in vitro DNA binding assay, we refine the features of motifs and flanking sequences that are critical for DCC binding. Our work reveals general principles by which regulatory complexes can be recruited across an entire chromosome to control its gene expression.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Mecanismo Genético de Compensação de Dose , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Análise por Conglomerados , Motivos de Nucleotídeos , Cromossomo X/genética , Cromossomo X/metabolismoRESUMO
The coordinated regulation of gene expression at the transcriptional level is fundamental to development and homeostasis. Inducible systems are invaluable when studying transcription because the regulatory process can be triggered instantaneously, allowing the tracking of ordered mechanistic events. Here, we use precision run-on sequencing (PRO-seq) to examine the genome-wide heat shock (HS) response in Drosophila and the function of two key transcription factors on the immediate transcription activation or repression of all genes regulated by HS. We identify the primary HS response genes and the rate-limiting steps in the transcription cycle that GAGA-associated factor (GAF) and HS factor (HSF) regulate. We demonstrate that GAF acts upstream of promoter-proximally paused RNA polymerase II (Pol II) formation (likely at the step of chromatin opening) and that GAF-facilitated Pol II pausing is critical for HS activation. In contrast, HSF is dispensable for establishing or maintaining Pol II pausing but is critical for the release of paused Pol II into the gene body at a subset of highly activated genes. Additionally, HSF has no detectable role in the rapid HS repression of thousands of genes.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/genética , Regulação da Expressão Gênica/genética , Estresse Fisiológico/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , DNA Polimerase II/metabolismo , Proteínas de Ligação a DNA/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Fatores de Transcrição de Choque Térmico , Regiões Promotoras Genéticas/genética , Interferência de RNA , Fatores de Transcrição/genéticaRESUMO
Previous studies have shown that GAGA Factor (GAF) is enriched on promoters with paused RNA Polymerase II (Pol II), but its genome-wide function and mechanism of action remain largely uncharacterized. We assayed the levels of transcriptionally-engaged polymerase using global run-on sequencing (GRO-seq) in control and GAF-RNAi Drosophila S2 cells and found promoter-proximal polymerase was significantly reduced on a large subset of paused promoters where GAF occupancy was reduced by knock down. These promoters show a dramatic increase in nucleosome occupancy upon GAF depletion. These results, in conjunction with previous studies showing that GAF directly interacts with nucleosome remodelers, strongly support a model where GAF directs nucleosome displacement at the promoter and thereby allows the entry Pol II to the promoter and pause sites. This action of GAF on nucleosomes is at least partially independent of paused Pol II because intergenic GAF binding sites with little or no Pol II also show GAF-dependent nucleosome displacement. In addition, the insulator factor BEAF, the BEAF-interacting protein Chriz, and the transcription factor M1BP are strikingly enriched on those GAF-associated genes where pausing is unaffected by knock down, suggesting insulators or the alternative promoter-associated factor M1BP protect a subset of GAF-bound paused genes from GAF knock-down effects. Thus, GAF binding at promoters can lead to the local displacement of nucleosomes, but this activity can be restricted or compensated for when insulator protein or M1BP complexes also reside at GAF bound promoters.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , RNA Polimerase II/genética , Fatores de Transcrição/genética , Transcrição Gênica , Animais , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Técnicas de Silenciamento de Genes , Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Drosophila contains one (dCDK12) and humans contain two (hCDK12 and hCDK13) proteins that are the closest evolutionary relatives of yeast Ctk1, the catalytic subunit of the major elongation-phase C-terminal repeat domain (CTD) kinase in Saccharomyces cerevisiae, CTDK-I. However, until now, neither CDK12 nor CDK13 has been demonstrated to be a bona fide CTD kinase. Using Drosophila, we demonstrate that dCDK12 (CG7597) is a transcription-associated CTD kinase, the ortholog of yCtk1. Fluorescence microscopy reveals that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNA polymerase II (RNAPII), but is distinct from that of P-TEFb (dCDK9 + dCyclin T). Chromatin immunoprecipitation (ChIP) experiments confirm that dCDK12 is present on the transcribed regions of active Drosophila genes. Compared with P-TEFb, dCDK12 amounts are lower at the 5' end and higher in the middle and at the 3' end of genes (both normalized to RNAPII). Appropriately, Drosophila dCDK12 purified from nuclear extracts manifests CTD kinase activity in vitro. Intriguingly, we find that cyclin K is associated with purified dCDK12, implicating it as the cyclin subunit of this CTD kinase. Most importantly, we demonstrate that RNAi knockdown of dCDK12 in S2 cells alters the phosphorylation state of the CTD, reducing its Ser2 phosphorylation levels. Similarly, in human HeLa cells, we show that hCDK13 purified from nuclear extracts displays CTD kinase activity in vitro, as anticipated. Also, we find that chimeric (yeast/human) versions of Ctk1 containing the kinase homology domains of hCDK12/13 (or hCDK9) are functional in yeast cells (and also in vitro); using this system, we show that a bur1(ts) mutant is rescued more efficiently by a hCDK9 chimera than by a hCDK13 chimera, suggesting the following orthology relationships: Bur1 â CDK9 and Ctk1 â CDK12/13. Finally, we show that siRNA knockdown of hCDK12 in HeLa cells results in alterations in the CTD phosphorylation state. Our findings demonstrate that metazoan CDK12 and CDK13 are CTD kinases, and that CDK12 is orthologous to yeast Ctk1.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Western Blotting , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular , Mapeamento Cromossômico , Ciclina T/genética , Ciclina T/metabolismo , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Teste de Complementação Genética , Células HeLa , Humanos , Microscopia de Fluorescência , Mutação , Fosforilação , Proteínas Quinases/genética , Interferência de RNA , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Transcriptional control plays a primary role in gene expression and can be exerted at multiple levels of the transcription process, such as RNA polymerase recruitment and promoter-proximal pausing. A recent report published in The EMBO Journal (Li and Gilmour, 2013) provides new insights into the regulation of a large class of paused genes through the identification of a novel transcription factor (TF; Motif 1 binding protein, M1BP) that binds to promoters of paused genes and controls their expression.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila/genética , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , TATA Box/genética , Fatores de Transcrição/genética , Transcrição Gênica , AnimaisRESUMO
In the eukaryotic genome, the thousands of genes that encode messenger RNA are transcribed by a molecular machine called RNA polymerase II. Analysing the distribution and status of RNA polymerase II across a genome has provided crucial insights into the long-standing mysteries of transcription and its regulation. These studies identify points in the transcription cycle where RNA polymerase II accumulates after encountering a rate-limiting step. When coupled with genome-wide mapping of transcription factors, these approaches identify key regulatory steps and factors and, importantly, provide an understanding of the mechanistic generalities, as well as the rich diversities, of gene regulation.
Assuntos
Regulação da Expressão Gênica , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Humanos , Cinética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/metabolismoRESUMO
Several eukaryotic transcription factors have been shown to modulate the elongation rate of RNA polymerase II (Pol II) on naked or chromatin-reconstituted templates in vitro. However, none of the tested factors have been shown to directly affect the elongation rate of Pol II in vivo. We performed a directed RNAi knock-down (KD) screen targeting 141 candidate transcription factors and identified multiple factors, including Spt6, that alter the induced Hsp70 transcript levels in Drosophila S2 cells. Spt6 is known to interact with both nucleosome structure and Pol II, and it has properties consistent with having a role in elongation. Here, ChIP assays of the first wave of Pol II after heat shock in S2 cells show that KD of Spt6 reduces the rate of Pol II elongation. Also, fluorescence recovery after photobleaching assays of GFP-Pol II in salivary gland cells show that this Spt6-dependent effect on elongation rate persists during steady-state-induced transcription, reducing the elongation rate from approximately 1100 to 500 bp/min. Furthermore, RNAi depletion of Spt6 reveals its broad requirement during different stages of development.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Fatores de Alongamento de Peptídeos/metabolismo , RNA Polimerase II/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Histonas/metabolismo , Fatores de Alongamento de Peptídeos/genética , Interferência de RNA , RNA Polimerase II/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
During M phase, Endosulfine (Endos) family proteins are phosphorylated by Greatwall kinase (Gwl), and the resultant pEndos inhibits the phosphatase PP2A-B55, which would otherwise prematurely reverse many CDK-driven phosphorylations. We show here that PP2A-B55 is the enzyme responsible for dephosphorylating pEndos during M phase exit. The kinetic parameters for PP2A-B55's action on pEndos are orders of magnitude lower than those for CDK-phosphorylated substrates, suggesting a simple model for PP2A-B55 regulation that we call inhibition by unfair competition. As the name suggests, during M phase PP2A-B55's attention is diverted to pEndos, which binds much more avidly and is dephosphorylated more slowly than other substrates. When Gwl is inactivated during the M phase-to-interphase transition, the dynamic balance changes: pEndos dephosphorylated by PP2A-B55 cannot be replaced, so the phosphatase can refocus its attention on CDK-phosphorylated substrates. This mechanism explains simultaneously how PP2A-B55 and Gwl together regulate pEndos, and how pEndos controls PP2A-B55. DOI: http://dx.doi.org/10.7554/eLife.01695.001.
Assuntos
Ciclo Celular , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/metabolismo , Regulação Enzimológica da Expressão Gênica , Peptídeos/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , FosforilaçãoRESUMO
Transcription regulation occurs frequently through promoter-associated pausing of RNA polymerase II (Pol II). We developed a precision nuclear run-on and sequencing (PRO-seq) assay to map the genome-wide distribution of transcriptionally engaged Pol II at base pair resolution. Pol II accumulates immediately downstream of promoters, at intron-exon junctions that are efficiently used for splicing, and over 3' polyadenylation sites. Focused analyses of promoters reveal that pausing is not fixed relative to initiation sites, nor is it specified directly by the position of a particular core promoter element or the first nucleosome. Core promoter elements function beyond initiation, and when optimally positioned they act collectively to dictate the position and strength of pausing. This "complex interaction" model was tested with insertional mutagenesis of the Drosophila Hsp70 core promoter.
Assuntos
Drosophila melanogaster/genética , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Iniciação da Transcrição Genética , Transcrição Gênica , Animais , Animais Geneticamente Modificados , Pareamento de Bases , Proteínas de Drosophila/genética , Regulação da Expressão Gênica , Genes de Insetos , Genoma de Inseto , Proteínas de Choque Térmico HSP70/genética , Modelos Genéticos , Mutagênese Insercional , Nucleossomos/metabolismo , Sítios de Splice de RNA , TransgenesRESUMO
Fcp1 dephosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (Pol II) to recycle it into a form that can initiate a new round of transcription. Previously, we identified Drosophila Fcp1 as an important factor in optimal Hsp70 mRNA accumulation after heat shock. Here, we examine the role of Fcp1 in transcription of heat shock genes in vivo. We demonstrate that Fcp1 localizes to active sites of transcription including the induced Hsp70 gene. The reduced Hsp70 mRNA accumulation seen by RNA interference (RNAi) depletion of Fcp1 in S2 cells is a result of a loss of Pol II in the coding region of highly transcribed heat shock-induced genes: Hsp70, Hsp26, and Hsp83. Moreover, Fcp1 depletion dramatically increases phosphorylation of the non-chromatin-bound Pol II. Reexpression of either wild-type or catalytically dead versions of Fcp1 demonstrates that both the reduced Pol II levels on heat shock genes and the increased levels of phosphorylated free Pol II are dependent on the catalytic activity of Fcp1. Our results indicate that Fcp1 is required to maintain the pool of initiation-competent unphosphorylated Pol II, and this function is particularly important for the highly transcribed heat shock genes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteínas de Choque Térmico HSP70/genética , Fosfoproteínas Fosfatases/metabolismo , RNA Polimerase II/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , Drosophila/genética , Proteínas de Drosophila/genética , Deleção de Genes , Proteínas de Choque Térmico/genética , Resposta ao Choque Térmico , Fosfoproteínas Fosfatases/genética , Fosforilação , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Estrutura Terciária de Proteína , Interferência de RNA , RNA Polimerase II/química , RNA Mensageiro/genéticaRESUMO
Positive transcription elongation factor b (P-TEFb) is the major metazoan RNA polymerase II (Pol II) carboxyl-terminal domain (CTD) Ser2 kinase, and its activity is believed to promote productive elongation and coupled RNA processing. Here, we demonstrate that P-TEFb is critical for the transition of Pol II into a mature transcription elongation complex in vivo. Within 3 min following P-TEFb inhibition, most polymerases were restricted to within 150 bp of the transcription initiation site of the active Drosophila melanogaster Hsp70 gene, and live-cell imaging demonstrated that these polymerases were stably associated. Polymerases already productively elongating at the time of P-TEFb inhibition, however, proceeded with elongation in the absence of active P-TEFb and cleared from the Hsp70 gene. Strikingly, all transcription factors tested (P-TEFb, Spt5, Spt6, and TFIIS) and RNA-processing factor CstF50 exited the body of the gene with kinetics indistinguishable from that of Pol II. An analysis of the phosphorylation state of Pol II upon the inhibition of P-TEFb also revealed no detectable CTD Ser2 phosphatase activity upstream of the Hsp70 polyadenylation site. In the continued presence of P-TEFb inhibitor, Pol II levels across the gene eventually recovered.