Saturday, 22 December 2012

Activation and repression

Activation functions 1 and 2 of nuclear receptors: molecular srategies for transcirptional activation by Warnmark et al

Nuclear receptors are ligand inducible TFs. They recruit general TFs to PIC or bind coactivators. NR binding to coactivators is mediated through activation domains, AF-1 and AF-2.

In a binding model, activation domain rapidly binds coactivator via weak electrostatic interactions. Complex slowly converts yo more stable form. Activation domain folds into defined structure.

The metazoan mediator coactivator compelx as an integrative hub for transcriptioanl regulation
Involvement of Mediator in multiple steps of transcription
Current models of transcriptional activation involve multiple steps. a | An idealized pathway is shown that begins with chromatin, in which nucleosomes exist in a characteristic beads-on-a-string array. This contrasts to transcriptionally inert chromatin, a highly compacted structure in which DNA is tightly packaged not just through wrapping around nucleosomes but through additional higher-order structures that entail linker histones and other proteins such as heterochromatin protein 1 (not shown). b | The activation pathway is initiated by one or more transcriptional activators that bind to their cognate sites in the regulatory region of the gene. These factors recruit a series of chromatin co-activators that can covalently modify nucleosomes at specific histone residues (not identified) and mobilize nucleosomes through ATP-requiring reactions. c | The resulting intermediate contains chromatin that is characterized by distinct covalent modifications, such as acetylation (Ac) and methylation (Me), and by a relative dearth of nucleosomes. The activators then recruit Mediator. In some cases, the intact Mediator that consists of the core and the kinase module might be recruited at this stage. d | Pre-initiation complex (PIC) assembly, entailing the various general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH) and RNA polymerase II (Pol II), and transcription initiation then ensue with concomitant restructuring of the Mediator that results from loss of the kinase module. e | After Pol II clears the promoter, there are two possible outcomes. As shown on the right, the process can proceed directly to the elongation phase at which it is associated with elongation factors that include DSIF and P-TEFb, whose entry into the transcription elongation complex (TEC) may coincide with capping (7-methyl-guanosine; 7 MeG) of the nascent RNA. The RPB1 carboxy-terminal domain (CTD) also undergoes substantial phosphorylation at Ser2 and Ser5 (Pol IIO) through the sequential actions of TFIIH and P-TEFb. Importantly, a scaffold containing a vestigial subset of general transcription factors and Mediator remains at the promoter to potentially facilitate subsequent rounds of transcription. Alternatively, as shown on the left, at many loci, Pol II may be subject to promoter-proximal pausing (at approximately nucleotide +50). This Pol II would be associated with DSIF but phosphorylated only at Ser 5 (Pol IIA). Under appropriate conditions, the paused Pol II complex can also mature into an elongation complex. Although depicted linearly, the intermediates are not likely to be as clear-cut as shown. Indeed, as discussed in the text, Mediator couples many of the steps. Moreover, some aspects of the reaction are reversible, again this occurs under the control of Mediator.
Analysis of Groucho-histone interactions suggest mechanistsic similarities between Groucho- and Tup-1-mediatred repression

Groucho gene (gro) encodes a tnrascriptional corepressor with homologs with all metazoanz. Gro lacks a DNA-binding domain. It is recruited to DNA by spec proitein-protein interactions with DNA-bound repressors. Gro is essnetial in develpomental processes eg segmentation, dorsal/ventral and terminal pattern formtaion, neurogenesis, sex determination and patterning of compound eye.

Gro interacts preferentially with hypoacetylated histones.  A western blot using Abs that recognise acetlated forms of histones H3 and H4 shows that no acetylated protein is detected antiacetylated H3 or H4.

N-terminal region of Gro excluding WD-repeat domain is required for efficient binding to core histones. Use GST pulldown assay.

Figure 6. A speculative model for Gro-mediated repression. (A) Multiple transcription factors can individually or synergistically recruit Gro to the DNA template. Gro in turn recruits a histone deacetylase activity. (B) Polymerization of Gro along the chromatin template facilitated by interactions with deacetylated histones allows the establishment of a large transcriptionally-silent domain.
Gro recruitment with HDAC could cause local deacetylation of template. Recruits more Gro, enlarging Gro-domain. This generates a large transcriptionally silent domain. Binding of Gro to histone tails could inhibit activity of HATs, maintaing hypoacetylated state associated with repression.

Histone modifications: now summoning sumoylation
Sumoylation stimulates activity eg HSF1 and tumour suppressor p53/.  It correlates with decreased transcription and repression of target genes.

H4 histone is sumoylated. Expression of reporter is decreased by targting EBC9 which conjugates sUMO to its substrate.

SUMO-H4 associates with chromatin.

Gene activation correlates with acetlation by HATs. Once gene is transcribed, its additivity must be attenuated and repressed. Signal for recruitment of sumoylating enzymes may be acetylation.  H4 sumpoylation increases with increasing H4 acetylation. HDAC mediates removal of acetyl groups recruited by DNA bound repressors. Repression by histone methyltransferase methylation. Required for HP1 binding which enables chromatin condensation.

ig. 1.
Model for sumoylation function in transcription. Horizontal line represents a gene with a TATA box-containing promoter and ORF; ovals represent histone octamers/nucleosomes. Through a coactivator, a DNA-bound activator can recruit a histone acetyltransferase (HAT) that acetylates histones and promotes chromatin structure amenable to transcription. This acetylation can potentially recruit SUMO-conjugating enzymes (E2/E3) capable of modifying either histones or activators to give an attenuating effect. A corepressor and HDAC activity could then be recruited by a DNA-bound repressor (possibly even with SUMO contributing to the interaction), deacetylating histones, and making way for the addition of repression-specific methylation marks, such as H3 K9-methyl, by an HMT. Finally, methylated histones (and possibly SUMO) would recruit HP1, contributing to chromatin structure in a static repressed state.





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