During which stage of transcription does rna polymerase bind to the promoter?

Catabolite Activator Protein

Transcription activation is a process carried out by a combination of a complex set of gene activators. The catabolite activator protein (CAP, also known as cAMP receptor protein, CRP) is a transcriptional activator, present as homodimer in solution, each subunit including a ligand-binding domain at the N-terminus and a DNA-binding domain at the C-terminus.82 Two cAMP molecules bind dimeric CAP and function as allosteric effectors by increasing the affinity for DNA. Components of the CAP superfamily have been linked to several biological functions, including immune defense. Many components contain a CAP domain, whereas others contain additional C-terminus extensions related to biological roles. A differential screening between LPS-treated and naïve C. intestinalis allowed the isolation of a full-length cDNA. Analysis of the deduced amino acid sequence showed that the protein (CiCAP) displays a modular structure with similarities to the vertebrate CAP superfamily as well as to a collagen-binding adhesin of Streptococcus mutans.83 Quantitative mRNA expression, performed by real-time PCR analysis, showed that the gene transcription is promptly activated in the pharynx after LPS inoculation. Moreover, in situ hybridization assay disclosed that CiCAP mRNA is highly produced by hemocytes with large granules that are contained inside the pharynx vessels. Thus CiCAP represents a protein with novel structural domains, involved in ascidian immune responses, probably as a component of the transcription activation complex.

1.1.1.1.2.1 Location in the Epigenome

Transcriptional activation or inactivation of specific miRNA genes is largely influenced by epigenetics. Such epigenetic regulation includes the proximity of the miRNA gene promoter to a CpG island, various histone modifications to the chromatin, and availability of factors that maintain and regulate expression from the epigenome. The expression of mir-127, a miRNA located near a CpG island is dependent on the methylation status of the promoter, implying epigenetic control on the expression of miRNAs (Lujambio, Ropero, Ballestar, et al., 2007; Saito, Liang, Egger, et al., 2006). MiRNAs also undergo massive upregulation when the DNA methylatransferases 1 and 3b (DNMT1, DNMT3) are downregulated (Lujambio et al., 2007), lending further support to the role of DNA methylation in regulating miRNA expression (reviews: Calin & Fabbri, 2010; Sato et al., 2011).

G Histone Modifications Modulate MPC Fate

Transcriptional activation and repression involves characteristic changes in the chromatin structure of regulated genes. Because key developmental regulators often bind to thousands of genes (e.g., Refs. 76,77), broad changes in chromatin structure might be expected. Histone deacetylases (HDAC) are central players in the control of chromatin structure through their regulated activity that removes covalent acetyl-modifications from histones and other chromosomal proteins including DNA-binding TFs. Chemical inhibitors of HDACs, for example, can induce widespread gene activation. Surprisingly, in some developmental contexts, nonspecific inhibition of HDAC activity can favor one developmental lineage over another. In particular, treatment of embryonic pancreatic explants in long-term culture with HDAC inhibitors suppresses acinar development.78 In explants treated with the inhibitors, the number of cells that express the proendocrine TF Ngn3 increases several-fold and persists many days longer than normal. This observation suggests that HDAC activity normally biases the cell-fate options toward the acinar program by suppressing the opportunity to enter the bipotent intermediate toward islet or ductal fates (Fig. 3). A thorough analysis of histone marks and DNA methylation in acinar and nonacinar cells will provide a road map for the pancreatic epigenome.

Alternative Splicing

Transcription initiation is certainly the first point that comes to mind when chromatin structure and gene expression are related. However, control of splicing might be at least as important. Splicing is executed by the trans-acting splicing factors that remove introns from pre-mRNA and fuse the remaining exons. A genome-wide survey of nucleosome positioning data in human T cells discovered that nucleosome density is higher over exons than over introns (Schones et al., 2008). Presumably, pausing of polymerase II (Pol II) at exons allows more time for the splicing machinery to recognize exons and splice out introns. Later, H3K36me3, H3K27me, H3K79me1/2, H4K20me1, and H2BK5me1 were found to be enriched in exons (reviewed in Zhou et al., 2014). Alternate splicing is one of the “hallmarks” of cancer progression (Oltean and Bates, 2014) and the existence of specific cancer isoforms is of diagnostic and eventually therapeutic interest. Mutations and dysregulation of expression of splice factors in cancer (Sveen et al., 2016) are confounding with a potential impact of cis-acting epigenetic modifications. However, both can and probably will contribute to aberrant splicing in tumor cells.

Initiation

Transcription initiation begins with formation of the pre-initiation complex (PIC) on promoter DNA. The PIC contains Pol II and the general transcription factors TFIIB, -D, -E, -F, and -H. The general factors are involved in sequence-specific promoter recognition (TFIIB and TFIID), prevention of nonspecific DNA binding (TFIIF), and DNA melting and CTD phosphorylation (TFIIE and TFIIH). Recently, the structure of RNA Pol II in complex with TFIIB was presented. The structure shows that to initiate gene transcription, promoter DNA is positioned over the Pol II active center cleft with the B-core domain that binds the wall at the end of the cleft. DNA is then opened with the help of the B-linker that binds the Pol II rudder and clamp coiled coil at the edge of the cleft. The DNA template strand slips into the cleft and is scanned for the transcription start site with the help of the B-reader that approaches the active site. RNA synthesis initiates within the bubble. The early transcribing complex is functionally unstable. Short RNAs are frequently released and Pol II has to restart transcription (abortive cycling).

Synthesis of the RNA chain and rewinding of upstream DNA displace the B-reader and B-linker, respectively, to trigger TFIIB release and EC formation. The elongating RNA extends over the dock domain, preventing reassociation of the TFIIB ribbon with Pol II during elongation.

Abstract

Transcriptional activation of proapoptotic genes play an important role to initiate cell death during animal development or in response to cytotoxic stress. In particular, P53-mediated induction of proapoptotic genes serves as a major tumor suppression mechanism in eliminating genetically compromised cells. During animal development, the sensitivity of cells to intra- and extracellular stress-induced cell death varies greatly depending on tissue types and differentiation status. Emerging evidences from Drosophila, as well as mammalian systems, indicated that targeted epigenetic regulation of proapoptotic genes, especially those that are transcriptional targets of P53, controls cellular sensitivity to stress-induced apoptosis during animal development. Epigenetic regulation of proapoptotic genes distinguishes itself from canonical epigenetic regulation of homeotic genes in three major aspects. First, it is not strictly determined by cell lineage or position. Second, it is often reversible in response to changes of the extracellular and intracellular environment. Last, but probably most importantly, epigenetic regulation of P53 target loci introduces differential sensitivity to stress among adjacent cells in the same tissue compartment. This phenotypic plasticity generated by the epigenetic mechanism may play an import role in maintaining appropriate number of cells during animal development. The detailed mechanism of how stress-responsive proapoptotic genes is specifically targeted for epigenetic suppression remains to be explored. However, abundant evidence suggests that such a mechanism can be hijacked by oncogenic viruses or dysregulated by oncogene overexpression to promote tumorigenesis. Comprehensive understanding of epigenetic regulation of proapoptotic genes holds the promise of improving the efficacy of cancer therapy.

3.2 Epigenetic Pathways

Transcription activation by ER is known to involve several coactivators. These coactivators have acetyltransferase activity and are capable of acetylating histones, which destabilizes nucleosomes and promotes transcription. Acetylation is regulated in a steady state by the enzymes histone acetyltransferase (HAT) and histone deacetylases (HDACs) activity.34 Expression of ER can be lost by epigenetic modifıcations such as histone deacetylation, which can cause endocrine resistance.

Pfister et al. demonstrated a correlation between histone modification and tumor phenotypes as well as patient outcomes. Higher levels of acetylation and methylation were associated with better outcomes compared with moderate or low levels.35

There is also some evidence of partial restoration of functional ER in cells that have lost ER expression as a result of acquired resistance to endocrine therapy.36

HDAC inhibitors (HDACi) were shown to induce growth arrest, differentiation, and cell death in breast cancer cells. HDACi inhibit ERα expression and cell proliferation. In breast cancer cells, TSA, which is a potent and reversible HDACi, led to a significant reduction in ERα accumulation independent of ER ligands. In human cancer cells, inhibition of HDACs regulates the expression of the ERα gene and the transcriptional activity in response to partial antiestrogens. Inhibition of HDAC enzymatic activity and modulation of ERα levels affect the relative agonist activity of partial antiestrogens on a stably integrated reporter transgene.34

Clinically the combination of vorinostat, a HDACi, and tamoxifen was shown to reverse hormone resistance, with a response rate of 19% and a clinical benefit rate of 40%.37

Epigenetic pathways therefore play a significant role in endocrine resistance and provide a potential therapeutic target.

Transcriptional Activation of Growth Factor Receptor Genes

Transcriptional activation is one of the means of regulating gene expression in many cell types and is governed by complexes of transcription factors. The lack of consensus TATA- and CCAAT-boxes in the majority of analyzed growth factor receptor gene promoters suggests that other DNA-binding sites present in the promoter region and their interactions with site-specific transcription factors are required for basal as well as heightened transcriptional activity. For the few growth factor receptor promoters that do contain consensus CCAAT-boxes, most notably PDGFRα and PDGFRβ, these sites bind positive transcription factors. The PDGFRα CCAAT element binds the CCAAT/enhancer-binding protein δ and the TGF-βR CCAAT element binds the nuclear factor Y (NFY) transcription factor. Numerous studies have implicated Sp transcription factors in the positive regulation of promoter activities for these growth factor receptor genes. It has been proposed that such multiple Sp-binding sites and associated protein(s) may stabilize the transcriptional machinery and establish a site of transcription initiation in promoters without TATA elements. Sp1 activates the promoters of TGF-βRI, TGF-βRII, TGF-βRIII, FGFR1–4, IGF-IR, EGFR, and HGFR. Most of these promoters contain 1 to 11 Sp sites in their regulatory region.

Positive transcriptional activity is conferred to the FGFR1 promoter during skeletal myoblast proliferation. Occupancy of an increasing number of Sp sites in the FGFR1 promoter additively increases transcriptional activity. However, the order of Sp-binding sites in relation to the start of transcription does not necessarily correlate with activity in regulating transcription. The chicken FGFR1 promoter contains three Sp sites in the proximal region, between −60 to −23 bp from the start of transcription. Mutation in the most proximal region (−23 Sp site) abolished promoter activity, indicating that this site confers most of the promoter activity. Mutation of the other two sites also reduced promoter activity to a lesser extent. Moreover, two additional Sp sites located 1 kb upstream from these proximal sites are also required for transcriptional activity of the FGFR1 gene promoter in proliferating myoblasts. These results suggest that Sp1–Sp1 protein interactions may induce looping of the intervening promoter sequences to create protein–protein complexes formed by distant cis-elements.

Other members of the Sp family, such as Sp2, Sp3, and Sp4, are also involved in the regulation of growth factor receptor genes. Like Sp1, both Sp3 and Sp4 can bind to GC-box or GT motifs, whereas Sp2 has a much weaker binding affinity for GT motifs. Sp2 and Sp4 often function as transcription activators, whereas the function of Sp3 is highly variable. Sp3 is a bifunctional transcription factor that can either activate or repress transcription. It has discrete domains for both activation and repression and therefore either activates or represses gene transcription depending on the cellular context. For instance, Sp3 augmented Sp1-mediated transcription of the FGFR1 gene promoter in chicken myoblasts. However, Sp3 itself was not able to activate the FGFR1 promoter. Therefore, Sp3 may function as a transcriptional coactivator of growth factor receptor promoters, in particular molecular and cellular contexts defined by the presence and activity of other interacting activators.

In many cases, Sp1 interacts with transcriptional factors other than Sp family members and synergistically activates growth factor receptor gene expression. The mouse TGF-βRIII promoter has been cloned and its sequence showed putative binding sites for Sp1, Smad3, Smad4, myogenic transcriptional regulators such as MyoD and myocyte enhancer factor 2 (MEF2), and retinoic acid receptor within a 2100 bp segment. All these factors are involved in the up-regulation of TGF-βRII gene expression in the mouse myogenic cell line, C2C12.

Other transcription factors are also involved in the transcriptional activation of growth factor receptor genes and they are mostly found in cancer cells. For instance, the murine TGF-βRII gene promoter contains two conserved Ets-binding sites and mE1f-3, a member of the Ets family, plays a key role in the activation of the promoter in mouse embryonal carcinoma cells. Similarly, AP-1 binds to at least seven sites in the EGFR promoter region and activates expression of the EGFR gene, which may contribute to cancer cell progression. Transcription factor Pax1 also acts as a transcriptional activator of the PDGFRα gene in differentiated Tera-2 human embryonic carcinoma cells. Many growth factor receptor gene promoters have been analyzed for interactions with Sp1 by site-specific binding assays and functional assays of expressed protein. However, the functional importance of transcription factors other than Sp factors binding to these promoters is poorly understood.

What stage involves RNA polymerase binding to a promoter sequence?

Does RNA polymerase bind to promoter?

Where does RNA polymerase bind and begin transcription?

Where does RNA polymerase bind?