Stimulate Transcription Initiation Research Articles

Building regulatory landscapes reveals that an enhancer can recruit cohesin to create contact domains, engage CTCF sites and activate distant genes

Developmental gene expression is often controlled by distal regulatory DNA elements called enhancers. Distant enhancer action is restricted to structural chromosomal domains that are flanked by CTCF-associated boundaries and formed through cohesin chromatin loop extrusion. To better understand how enhancers, genes and CTCF boundaries together form structural domains and control expression, we used a bottom-up approach, building series of active regulatory landscapes in inactive chromatin. We demonstrate here that gene transcription levels and activity over time reduce with increased enhancer distance. The enhancer recruits cohesin to stimulate domain formation and engage flanking CTCF sites in loop formation. It requires cohesin exclusively for the activation of distant genes, not of proximal genes, with nearby CTCF boundaries supporting efficient long-range enhancer action. Our work supports a dual activity model for enhancers: its classic role of stimulating transcription initiation and elongation from target gene promoters and a role of recruiting cohesin for the creation of chromosomal domains, the engagement of CTCF sites in chromatin looping and the activation of distal target genes.

Complex-dependent histone acetyltransferase activity of KAT8 determines its role in transcription and cellular homeostasis

Acetylation of lysine 16 on histone H4 (H4K16ac) is catalyzed by histone acetyltransferase KAT8 and can prevent chromatin compaction invitro. Although extensively studied in Drosophila, the functions of H4K16ac and two KAT8-containing protein complexes (NSL and MSL) are not well understood in mammals. Here, we demonstrate a surprising complex-dependent activity of KAT8: it catalyzes H4K5ac and H4K8ac as part of the NSL complex, whereas it catalyzes the bulk of H4K16ac as part of the MSL complex. Furthermore, we show that MSL complex proteins and H4K16ac are not required for cell proliferation and chromatin accessibility, whereas the NSL complex is essential for cell survival, as it stimulates transcription initiation at the promoters of housekeeping genes. In summary, we show that KAT8 switches catalytic activity and function depending on its associated proteins and that, when in the NSL complex, it catalyzes H4K5ac and H4K8ac required for the expression of essential genes.

Distinct roles of nucleosome sliding and histone modifications in controlling the fidelity of transcription initiation

ABSTRACT Regulation of gene expression starts from the transcription initiation. Regulated transcription initiation is critical for generating correct transcripts with proper abundance. The impact of epigenetic control, such as histone modifications and chromatin remodelling, on gene regulation has been extensively investigated, but their specific role in regulating transcription initiation is far from well understood. Here we aimed to better understand the roles of genes involved in histone H3 methylations and chromatin remodelling on the regulation of transcription initiation at a genome-scale using the budding yeast as a study system. We obtained and compared maps of transcription start site (TSS) at single-nucleotide resolution by nAnT-iCAGE for a strain with depletion of MINC (Mot1-Ino80C-Nc2) by Mot1p and Ino80p anchor-away (Mot1&Ino80AA) and a strain with loss of histone methylation (set1Δset2Δdot1Δ) to their wild-type controls. Our study showed that the depletion of MINC stimulated transcription initiation from many new sites flanking the dominant TSS of genes, while the loss of histone methylation generates more TSSs in the coding region. Moreover, the depletion of MINC led to less confined boundaries of TSS clusters (TCs) and resulted in broader core promoters, and such patterns are not present in the ssdΔ mutant. Our data also exhibits that the MINC has distinctive impacts on TATA-containing and TATA-less promoters. In conclusion, our study shows that MINC is required for accurate identification of bona fide TSSs, particularly in TATA-containing promoters, and histone methylation contributes to the repression of transcription initiation in coding regions.

The Cdk8 kinase module regulates interaction of the mediator complex with RNA polymerase II

The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex mediator, which regulates gene transcription by RNA polymerase II. The CKM has both negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. In order to reconstitute the role of the CKM in transcription initiation, we prepared recombinant CKM from the yeast Saccharomyces cerevisiae. We showed that CKM bound to the core mediator (cMed) complex, sterically inhibiting cMed from binding to the polymerase II preinitiation complex (PIC) in vitro. We further showed that the Cdk8 kinase activity of the CKM weakened CKM–cMed interaction, thereby facilitating dissociation of the CKM and enabling mediator to bind the PIC in order to stimulate transcription initiation. Finally, we report that the kinase activity of Cdk8 is required for gene activation during the stressful condition of heat shock in vivo but not under steady-state growth conditions. Based on these results, we propose a model in which the CKM negatively regulates mediator function at upstream-activating sequences by preventing mediator binding to the PIC at the gene promoter. However, during gene activation in response to stress, the Cdk8 kinase activity of the CKM may release mediator and allow its binding to the PIC, thereby accounting for the positive function of CKM. This may impart improved adaptability to stress by allowing a rapid transcriptional response to environmental changes, and we speculate that a similar mechanism in metazoans may allow the precise timing of developmental transcription programs.

Sequence-Directed Action of RSC Remodeler and General Regulatory Factors Modulates +1 Nucleosome Position to Facilitate Transcription

Accessible chromatin is important for RNA polymerase II recruitment and transcription initiation at eukaryotic promoters. We investigated the mechanistic links between promoter DNA sequence, nucleosome positioning, and transcription. Our results indicate that positioning of the transcription start site-associated+1 nucleosome in yeast is critical for efficient TBP binding and is driven by two key factors, the essential chromatin remodeler RSC and a small set of ubiquitous general regulatory factors (GRFs). Our findings indicate that the strength and directionality of RSC action on promoter nucleosomes depends on the arrangement and proximity of two specific DNA motifs. This, together with the effect on nucleosome position observed in double depletion experiments, suggests that, despite their widespread co-localization, RSC and GRFs predominantly act through independent signals to generate accessible chromatin. Our results provide mechanistic insight into how the promoter DNA sequence instructs trans-acting factors to control nucleosome architecture and stimulate transcription initiation.

The Eaf3/5/7 Subcomplex Stimulates NuA4 Interaction with Methylated Histone H3 Lys-36 and RNA Polymerase II

NuA4 is the only essential lysine acetyltransferase complex in Saccharomyces cerevisiae, where it has been shown to stimulate transcription initiation and elongation. Interaction with nucleosomes is stimulated by histone H3 Lys-4 and Lys-36 methylation, but the mechanism of this interaction is unknown. Eaf3, Eaf5, and Eaf7 form a subcomplex within NuA4 that may also function independently of the lysine acetyltransferase complex. The Eaf3/5/7 complex and the Rpd3C(S) histone deacetylase complex have both been shown to bind di- and trimethylated histone H3 Lys-36 stimulated by Eaf3. We investigated the role of the Eaf3/5/7 subcomplex in NuA4 binding to nucleosomes. Different phenotypes of eaf3/5/7Δ mutants support functions for the complex as both part of and independent of NuA4. Further evidence for Eaf3/5/7 within NuA4 came from mutations in the subcomplex leading to ∼40% reductions in H4 acetylation in bulk histones, probably caused by binding defects to both nucleosomes and RNA polymerase II. In vitro binding assays showed that Eaf3/5/7 specifically stimulates NuA4 binding to di- and trimethylated histone H3 Lys-36 and that this binding is important for NuA4 occupancy in transcribed ORFs. Consistent with the role of NuA4 in stimulating transcription elongation, loss of EAF5 or EAF7 resulted in a processivity defect. Overall, these results reveal the function of Eaf3/5/7 within NuA4 to be important for both NuA4 and RNA polymerase II binding.

SaRNA-guided Ago2 targets the RITA complex to promoters to stimulate transcription.

Small activating RNAs (saRNAs) targeting specific promoter regions are able to stimulate gene expression at the transcriptional level, a phenomenon known as RNA activation (RNAa). It is known that RNAa depends on Ago2 and is associated with epigenetic changes at the target promoters. However, the precise molecular mechanism of RNAa remains elusive. Using human CDKN1A (p21) as a model gene, we characterized the molecular nature of RNAa. We show that saRNAs guide Ago2 to and associate with target promoters. saRNA-loaded Ago2 facilitates the assembly of an RNA-induced transcriptional activation (RITA) complex, which, in addition to saRNA-Ago2 complex, includes RHA and CTR9, the latter being a component of the PAF1 complex. RITA interacts with RNA polymerase II to stimulate transcription initiation and productive elongation, accompanied by monoubiquitination of histone 2B. Our results establish the existence of a cellular RNA-guided genome-targeting and transcriptional activation mechanism and provide important new mechanistic insights into the RNAa process.

HMBA Enhances Prostratin-Induced Activation of Latent HIV-1 via Suppressing the Expression of Negative Feedback Regulator A20/TNFAIP3 in NF-κB Signaling.

In the past decade, much emphasis has been put on the transcriptional activation of HIV-1, which is proposed as a promised strategy for eradicating latent HIV-1 provirus. Two drugs, prostratin and hexamethylene bisacetamide (HMBA), have shown potent effects as inducers for releasing HIV-1 latency when used alone or in combination, although their cellular target(s) are currently not well understood, especially under drug combination. Here, we have shown that HMBA and prostratin synergistically release HIV-1 latency via different mechanisms. While prostratin strongly stimulates HMBA-induced HIV-1 transcription via improved P-TEFb activation, HMBA is capable of boosting NF-κB-dependent transcription initiation by suppressing prostratin-induced expression of the deubiquitinase A20, a negative feedback regulator in the NF-κB signaling pathway. In addition, HMBA was able to increase prostratin-induced phosphorylation and degradation of NF-κB inhibitor IκBα, thereby enhancing and prolonging prostratin-induced nuclear translocation of NF-κB, a prerequisite for stimulation of transcription initiation. Thus, by blocking the negative feedback circuit, HMBA functions as a signaling enhancer of the NF-κB signaling pathway.

Abscisic acid affects transcription of chloroplast genes via protein phosphatase 2C-dependent activation of nuclear genes: repression by guanosine-3'-5'-bisdiphosphate and activation by sigma factor 5.

Abscisic acid (ABA) represses the transcriptional activity of chloroplast genes (determined by run-on assays), with the exception of psbD and a few other genes in wild-type Arabidopsis seedlings and mature rosette leaves. Abscisic acid does not influence chloroplast transcription in the mutant lines abi1-1 and abi2-1 with constitutive protein phosphatase 2C (PP2C) activity, suggesting that ABA affects chloroplast gene activity by binding to the pyrabactin resistance (PYR)/PYR1-like or regulatory component of ABA receptor protein family (PYR/PYL/RCAR) and signaling via PP2Cs and sucrose non-fermenting protein-related kinases 2 (SnRK2s). Further we show by quantitative PCR that ABA enhances the transcript levels of RSH2, RSH3, PTF1 and SIG5. RelA/SpoT homolog 2 (RSH2) and RSH3 are known to synthesize guanosine-3'-5'-bisdiphosphate (ppGpp), an inhibitor of the plastid-gene-encoded chloroplast RNA polymerase. We propose, therefore, that ABA leads to an inhibition of chloroplast gene expression via stimulation of ppGpp synthesis. On the other hand, sigma factor 5 (SIG5) and plastid transcription factor 1 (PTF1) are known to be necessary for the transcription of psbD from a specific light- and stress-induced promoter (the blue light responsive promoter, BLRP). We demonstrate that ABA activates the psbD gene by stimulation of transcription initiation at BLRP. Taken together, our data suggest that ABA affects the transcription of chloroplast genes by a PP2C-dependent activation of nuclear genes encoding proteins involved in chloroplast transcription.

MYC and Transcription Elongation

Most transcription factors specify the subset of genes that will be actively transcribed in the cell by stimulating transcription initiation at these genes, but MYC has a fundamentally different role. MYC binds E-box sites in the promoters of active genes and stimulates recruitment of the elongation factor P-TEFb and thus transcription elongation. Consequently, rather than specifying the set of genes that will be transcribed in any particular cell, MYC's predominant role is to increase the production of transcripts from active genes. This increase in the transcriptional output of the cell's existing gene expression program, called transcriptional amplification, has a profound effect on proliferation and other behaviors of a broad range of cells. Transcriptional amplification may reduce rate-limiting constraints for tumor cell proliferation and explain MYC's broad oncogenic activity among diverse tissues.

Gene Expression Is Circular: Factors for mRNA Degradation Also Foster mRNA Synthesis

Maintaining proper mRNA levels is a key aspect in the regulation of gene expression. The balance between mRNA synthesis and decay determines these levels. We demonstrate that most yeast mRNAs are degraded by the cytoplasmic 5'-to-3' pathway (the "decaysome"), as proposed previously. Unexpectedly, the level of these mRNAs is highly robust to perturbations in this major pathway because defects in various decaysome components lead to transcription downregulation. Moreover, these components shuttle between the cytoplasm and the nucleus, in a manner dependent on proper mRNA degradation. In the nucleus, they associate with chromatin-preferentially ∼30 bp upstream of transcription start-sites-and directly stimulate transcription initiation and elongation. The nuclear role of the decaysome in transcription is linked to its cytoplasmic role in mRNA decay; linkage, in turn, seems to depend on proper shuttling of its components. The gene expression process is therefore circular, whereby the hitherto first and last stages are interconnected.

Single Molecule Probing of the Human Tumor Suppression Transcription Initiation Machinery

In humans, transcription Pre-Initiation Complex (PIC) assembly begins with the recognition of the core promoter by the large multi-subunit TFIID complex and culminates with recruitment of RNA Polymerase II. Transcriptional activators, such as the tumor suppressor p53 protein, stimulate transcription initiation, in part by binding sequences upstream of the promoter and help recruit TFIID to core promoter DNA.In response to cellular stress the tumor suppressor p53 protein becomes activated and turns on a variety of target genes in a highly coordinated manner to dictate cellular fate. Cellular levels of p53 are essential in dictating gene expression programs based upon the affinity of p53 for binding sites upstream of the core promoter. Many general transcription factors that bind to the core promoter are known to interact with p53 and potentially alter the affinity of each factor for the promoter DNA. Despite almost 20 years of research, the dynamic interplay between p53 and components within the general transcription machinery at physiological promoters remains poorly understood.We have probed in real-time the association between human p53, TFIID, and various p53 target gene promoters, using a combination of in vitro single molecule tethered particle motion(TPM) and high-resolution TIRF co-localization assays. Our results show that p53 and TFIID act cooperatively to aid in recruitment to promoter DNA. In addition, differences in cooperative assembly of p53 and TFIID on various physiological promoters underscore a mechanism for p53's graded response to cellular stress. TPM analysis reveals p53 and TFIID dependent DNA looping suggesting stable interactions between upstream bound activators and core promoter bound factors. We have further examined the influence of additional general transcription factors on p53 mediated transcription complex assembly.

Crosstalk between mRNA 3′ End Processing and Transcription Initiation

Transcription and mRNA maturation are interdependent events. Although stimulatory connections between these processes within the same round of transcription are well described, functional coupling between separate transcription cycles remains elusive. Comparing time-resolved transcription profiles of single-copy integrated β-globin gene variants, we demonstrate that a polyadenylation site mutation decreases transcription initiation of the same gene. Upon depletion of the 3' end processing and transcription termination factor PCF11, endogenous genes exhibit a similar phenotype. Readthrough RNA polymerase II (RNAPII) engaged on polyadenylation site-mutated transcription units sequester the transcription initiation/elongation factors TBP, TFIIB and CDK9, leading to their depletion at the promoter. Additionally, high levels of TBP and TFIIB appear inside the gene body, and Ser2-phosphorylated RNAPII accumulates at the promoter. Our data demonstrate that 3' end formation stimulates transcription initiation and suggest that coordinated recycling of factors from a gene terminator back to the promoter is essential for sustaining continued transcription.

NuA4 Lysine Acetyltransferase Esa1 Is Targeted to Coding Regions and Stimulates Transcription Elongation with Gcn5

NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4's interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.

Bicarbonate‐mediated transcriptional activation of divergent operons by the virulence regulatory protein, RegA, from Citrobacter rodentium

Regulation of virulence gene expression plays a central role in the pathogenesis of enteric bacteria as they encounter diverse environmental conditions in the gastrointestinal tract of their hosts. In this study, we investigated environmental regulation of two putative virulence determinants adcA and kfc by RegA, an AraC/XylS-like regulator, from Citrobacter rodentium, and identified bicarbonate as the environmental signal which induced transcription of adcA and kfc through RegA. Primer extension experiments showed that adcA and kfc were divergently transcribed from sigma(70) promoters. In vivo and in vitro experiments demonstrated that bicarbonate facilitated and stabilized the binding of RegA to an operator located between the two promoters. The interaction of RegA with its DNA target resulted in the formation of a nucleosome-like structure, which evidently displaced the histone-like proteins, H-NS and StpA, from the adcA and kfc promoter regions, leading to transcriptional derepression. In addition, our results indicated that RegA also behaved as a Class I activator by directly stimulating transcription initiation by RNA polymerase. This is the first report to describe the molecular mechanism by which an environmental chemical stimulates transcription of virulence-associated genes of an enteric pathogen through an AraC/XlyS-like activator.

A 5′ Splice Site Enhances the Recruitment of Basal Transcription Initiation Factors In Vivo

Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or beta-globin mRNAs, harboring wild-type or various 5' splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5' splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced promoter docking of transcription initiation factors TFIID, TFIIB, and TFIIH on a gene containing a functional 5' splice site. In addition to their promoter association, the TFIID and TFIIH components, TBP and p89, are specifically recruited to the 5' splice site region. Our data suggest a model in which a promoter-proximal 5' splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing pre-initiation complex assembly.

Mechanisms of physiological regulation of RNA synthesis in bacteria: new discoveries breaking old schemes

Although in bacterial cells all genes are transcribed by RNA polymerase, there are 2 additional enzymes capable of catalyzing RNA synthesis: poly(A) polymerase I, which adds poly(A) residues to transcripts, and primase, which produces primers for DNA replication. Mechanisms of actions of these 3 RNA-synthesizing enzymes were investigated for many years, and schemes of their regulations have been proposed and generally accepted. Nevertheless, recent discoveries indicated that apart from well-understood mechanisms, there are additional regulatory processes, beyond the established schemes, which allow bacterial cells to respond to changing environmental and physiological conditions. These newly discovered mechanisms, which are discussed in this review, include: (i) specific regulation of gene expression by RNA polyadenylation, (ii) control of DNA replication by interactions of the starvation alarmones, guanosine pentaphosphate and guanosine tetraphosphate, (p)ppGpp, with DnaG primase, (iii) a role for the DksA protein in ppGpp-mediated regulation of transcription, (iv) allosteric modulation of the RNA polymerase catalytic reaction by specific inhibitors of transcription, rifamycins, (v) stimulation of transcription initiation by proteins binding downstream of the promoter sequences, and (vi) promoter-dependent control of transcription antitermination efficiency.

A Sequence Motif in the Simian Virus 40 (SV40) Early Core Promoter Affects Alternative Splicing of Transcribed mRNA

To identify new sequence elements in the promoter that affect splicing patterns of pre-mRNAs, we analyzed effects of different promoters on alternative splicing of model reporter genes. We compared the E1a alternative splicing pattern in transcripts expressed from the full-length cytomegalovirus, SV40 early, or a hybrid cytomegalovirus/SV40 early promoter and found that the hybrid promoter improved selection of the suboptimal E1a 5'SS-1. Expressing RNA from the hybrid promoter also enhanced selection of suboptimal splice sites in other alternatively spliced reporter genes, demonstrating the generality of this effect. Unlike previously defined promoter elements shown to affect alternative splicing, which were located in the enhancer/upstream activating sequences, the motif identified in this work is positioned within the core promoter; it is comprised of eight T-residues directly upstream of the SV40 early TATA box. This motif was previously implicated in DNA bending and negative regulation of transcription. Together, these results suggest that the identity of transcription complex assembled in the core promoter-dependent fashion can affect splice site selection during pre-mRNA splicing, perhaps by influencing the processivity of transcription elongation.

Differential IFN-γ Stimulation of HLA-A Gene Expression through CRM-1-Dependent Nuclear RNA Export

IFNs regulate most MHC class I genes by stimulating transcription initiation. As shown previously, IFN-gamma controls HLA-A expression primarily at the posttranscriptional level. We have defined two 8-base sequences in a 39-nucleotide region in the 3'-transcribed region of the HLA-A gene that are required for the posttranscriptional response to IFN-gamma. Stimulation of HLA-A expression by IFN-gamma requires nuclear export of HLA-A mRNA by chromosome maintenance region 1 (CRM-1). Treatment of cells with leptomycin B, a specific inhibitor of CRM-1, completely inhibited IFN-gamma induction of HLA-A. Expression of a truncated, dominant-negative form of the nucleoporin NUP214/CAN, DeltaCAN, that specifically interacts with CRM-1, also prevented IFN-gamma stimulation of HLA-A, providing confirmation of the role of CRM-1. Increased expression of HLA-A induced by IFN-gamma also requires protein methylation, as shown by the fact that treatment of SK-N-MC cells or HeLa cells with the PRMT1 inhibitor 5'-methyl-5'-thioadenosine abolished the cellular response to IFN-gamma. In contrast with HLA-A, IFN-gamma-induced expression of the HLA class Ib gene, HLA-E, was not affected by either 5'-methyl-5'-thioadenosine or leptomycin B. These results provide proof of principle that it is possible to differentially modulate the IFN-gamma-induced expression of the HLA-E and HLA-A genes, whose products often mediate opposing effects on cellular immunity to tumor cells, pathogens, and autoantigens.

Direct Stimulation of Transcription Initiation by BRCA1 Requires Both Its Amino and Carboxyl Termini

Published experiments suggest that BRCA1 interaction with RNAPII and regulation of a number of target genes may be central to its role as a tumor suppressor. Previous in vivo and in vitro work has implicated the carboxyl terminus of BRCA1 in transcriptional stimulation, but the mechanism of action remains unknown, and whether the full-length protein stimulates transcription is controversial. BRCA1 interacts with a number of enhancer-binding transcriptional activators, suggesting that these factors recruit BRCA1 to promoters, where it stimulates RNA synthesis. To investigate whether BRCA1 has intrinsic transcriptional activity, we established a fully purified transcription assay. We demonstrate here that BRCA1 stimulates transcription initiation across a range of promoters. Both the amino and carboxyl termini of BRCA1 are required for this activity, but the BRCA1-binding partner, BARD1, is not. Our data support a model whereby BRCA1 stabilizes productive preinitiation complexes and thus stimulates transcription.