Saturday, 22 December 2012

Gene-specific transcription factors

Wet and dry interfaces: the role of solvent in protein-protein and protein-DNA recognition by Janin
Structural data on protein-protein and protein-DNA recognition sites indicate water is abundant at interfaces. Nearly all interface solvent molecules are involved in bridging H bonds. Protein-protein and protein- DNA interfaces have at least as many water-mediated interactions as direct H bonds or salt bridges. Water therefore plays a major role in polar interactions that stabilise complexes.

Protein-protein interfaces are as desnsely packed on average as interior of globular proteins. Water is almost fully excluded from interior. Few remaining cavities are empty. Like protein interior, a dry interface has few cavities. A wet interface has many cavities and all are filled with water to maintain close packing of atoms.

Average protein-protein interface is not much different in chemical composition from rest of protein surface. In contrast, protein- DNA interfaces are very particular. These interfaces are much more polar due to phosphate groups on DNA side and many positively charged groups on protein side.. This yields positive surface potential. However, dry and wet protein-DNA interfaces also exist.

Figure 2. Water molecules at protein–DNA interfaces. (a) The dimeric papilloma E2 protein in complex with DNA (PDB code 2bop[20](b) The human TATA-box-binding protein in complex with DNA (PDB code 1cdw[21]. The molecular surface of the protein is colored according to its electrostatic potential: blue, positive; red, negative. Red spheres represent water molecules reported in the PDB files and at a distance of less than 3.5 Å from atoms of both the protein and the DNA. (The figure was drawn using the program GRASP [19].)
In fig 2, water penetrates interface between papilloma E2 protein and DNA. DNA double helix is bent but is in B conformation. Contacts are mostly in major groove.

In contrast, water is excluded from central surface path of a DNA complex with TBP. Nonpolar contacts between protein side-chains and bases and sugars in minor groove of DNA. Large distortion of DNA double helix pushes phosphate backbone to edge of interface.

In general water molecules tend to follow phosphate backbone of DNA and H bond to phosphate oxygens but water mediated H bonds are also frequent.

Determining the specificity of protein-DNA interactions by Stormo and Zhao
One class of TFs interacts with DNA in a sequence- specific manner. It binds only locations in genome that contain appropriate DNA sequences. On binding, these TFs may regulate expression of associated genes, activating or repressing their RNA synthesis.

Many bindings sites in genome do not affect expression of nearby genes. TFs are a major determinant of regulated and controlled expression of all genes.

In vivo the affinity of TF is not as crucial as its specificity. In a bacterial cell or eukaryotic nucleus DNA concentration is so high that TFs will always bind DNA even without high affinity sites.

Crystal structure of λ repressor and a model for pairwise cooperative operator binding
Intact dimeric repressor bound to a specific 17bp operator sequence was cystallised.   NTD contains a HTH motif. It is a compact α-helical domain that weakly self-associates to form a dimer. NTD dimers recognise and bind operator using HTH.

The many faces of helix-turn-helix domain: transcription regulation and beyond by Aravind et al
Matthews et al showed that phage λ transcription regulators eg cro and cI repressor, and lacI the lactose operon repressor shared a similar trihelical DNA-binding domain. 2nd and 3rd helices constuituted a HTH motif. HTH domains have been discovered in eukaryotic transcirption factors, chromatin protein eg histone H1 and basal TFs eg TFIIB and TFIIE.

HTH is a fold of 3 core helices that form a right-handed helical bundle. The sharp turn between 2nd and 3rd helix does not tolerate insertions or distortions. Loop between helix 1 and 2 may accommodate modifications.  A shallow cleft between helix 1 and 2 may allow additional structural elements to pack into it via hydrophobic interactions. The 3rd helix is the recognition heliz. It forms the principal DNA-protien interface bny inserting into major groove of DNA.

Widely conserved seuqence elements eg shs (s is a small residue usually glycine in first position, h is hydrophobic residue) localise to interior ad form hydrophobic core that stabilises domain.

HTH domains with a simple three-helical bundle and its extensions
Simplest version of HTH domain is the basic trihelical cersion. It is 3 core helices with no elaborations.  Other versions include tetrahelical, multihelical and d winged HTH domain.

The crystal structure of an intact human Max-DNA complex: new insights into mechanisms of transcriptional control by Brownlie
X-ray structure of human Max protein homodimer in complex with 13-mer DNA duplex.  Max is a TF belonging to helix-loop-helix leucine zipper family (bHLHX) class on DNA-binding proteins. One member of the family, Myc, is an oncoprotein implicated in cell proliferation, differentiation and apoptosis. All known myc activities require interaction with Max.  . Resulting heterodimer activates transcription through trans-activating domain of Myc.  Myc-Max heterodimer binds CACGTG sequence (E box element).

DNA binding occurs through basic region of HLH motif. Both HLH and leucine zipper form part of dimerisation interface  in bHLHZ class of proteins. Max homodimers, as well as Max-Mad or Max-Mxi1 heterodimers bind same E box elements and act as transcriptional repressors.

Repression results from sequestration of Max (which prevents Myc-Max heterodimer formation) and from competition from common DNA target site.

Crystal structures of proteins with bHLH motif show high conservation. Conserved E box binding motif consists of a glutamate residue making specific base contacts.  Glutamate forms a salt bridge to an arginine side chain which forms a H bond with phosphate backbone.

Zinc fingers

Ribbon-helix-helix transcription factors: variations on a theme by Schreiter and Drennan
Functional unit of ribbon-helix-helix domain is a dimer (RHH2). 2 RHH motifs are tightly intertwined to form a stable domain with 2fold symmetry and can bind DNA. 2fold symmetry is usually formed by homodimersiation.  Paired short β strands at N terminus of each RHH monomre form an antiparallel β sheet. Binding of RHH2 domain to DNA positions this β sheet in major groove.  3 amino acid side chains from each strand point into groove and make drucual sequence-spec nutcleotide bas contacts.

Sequence-specific DNA binding by β strand residues is a defining characteristic of RHH superfamily. Distinguishes them from HTH superfamily, which insert an α helix into DNA major groove.

2 protein-backbone amide nitrogens at N terminus of 2nd α helix of RHH2 domain makes conserved set of nonspecific contacts to DNA phosphate backbone on either sied of major groove. This interaction is electrostatically favourable because positive dipole at helix N terminus is oriented directly towards a negatively charged phosphate of DNA backbone.
Three views of the RHH dimer (RHH2) domain are shown as cartoons and coloured by subunit. The three amino-acid positions (2, 4 and 6) from each subunit that make sequence-specific nucleotide base contacts are shown as semi-transparent spheres. a | Reference positions within the RHH motif are numbered on the green subunit. The numbers correspond directly to the sequence alignment in Figure 1. b | The secondary structure elements are labelled on the green subunit, which also corresponds to the alignment in Figure 1c | Shows the interaction of a RHH2 domain with a DNA operator. Nonspecific anchoring contacts between the N terminus of the second alpha-helix and the DNA phosphate backbone are shown as dashed black lines. Specific base contacts are made by positions 2, 4 and 6 of each subunit from the beta-sheet within the DNA major groove. All structural figures were prepared using PyMOL53.


E2F in vivo binding specificity: comparison of consensus versus nonconsensus binding sites by Rabinovich 
E2F family is implicated in controlling critical cellular function eg entrance in S phase, regulation of mitosis, apoptosis, DNA repair, DNA damage checkpoint control and organismal function.  In vitro bind consensus motif TTTSSGGC where S is either G or C.

Most sites bound by E2F family members in vivo do not contain E2F consensus motifs.  Using chromatin immnoprecipitation coupled with DNA microarray analysis, it is shown that prefominant factors specifying whether E2F is recruited to an invivo binding site are
1. site must be in a core promoter and
2. the region must be used as a promoter in that cell type.

3 Models for E2f recruitment to core promoters lacking a consensus site were tested, including
1. indirect recruitment
2. looping to core promoter mediated by an E2f bound to a distal motif
3. assisted binding of E2F to a site that weakly resembles an E2F motif

An in vivo assay, eChIP allows analysis of TF binding to isolateds fragments. In vivo a consensus motif is not suffiicient to recruit E2F. E2Fs can bind to isolated regions that lack a consensus motif. Binding can require regions other than best match to E2F motif.

Models for recruitment of E2F1 to promoters that lack a consensus motif. Shown are schematics representing potential modes of E2F1 recruitment via a mechanism independent of its DNA binding domain (A), via looping (B), or via stabilized binding in cooperation with another transcription factor (C).
An intact DNA-binding domain is required for binding to nonconsensus sites
E2F could be recruited to core promoter lacking consensus motif by protein-protein interactions with another factor in absence of direct DNA binding.

They created stable cell lines that expressed either wt E2F1 or E2F1 mutated in DNA-binding domain.  Constructs include HA tag and modified ER ligand binding domain to allow regulated translocation to nucleus. Nuclear extracts were prepared.

Comprae top-ranked promoters identified using HA antibody in HA-wtE2F1 cells to top ranked promoters identified using E2F1 antibody in parental (untransfected) MCF7 cells. 60% overlap in top ranked targets similar to overlap of many ChIP-chip replicates when same antibody is used with 2 independent cultures of cells. Thus, HA_tagged E2f1 (HA-wtE2F1) binds to same targets as endogenous E2F1.

ChIP-chip assays using HA tagged E2F1 DNA-binding domain mutant. Select set of top-ranked HA-wtE2F1 targets. Compare enrichment values at these promoters in ChIP assays that measured binding of endogenous E2F1, HA-wtE2F1 and HA-DBDmut E2F1 and enrichment using HA antibody in MCF7 cells harbouring only HA-ER vector plasmid.  Top 1000 promoters on HA-wtE2F1 array were bound with high enrichment valuesby endogenous and tagged wildtype.  DNA-binding domain mutant could not bind these E2F1 targets.

No evidence that E2F1 can be recruited to any promoter strictly by protein-protein interaction in absence of a functional DNA-binding domain.


E2F family members can bind promoters that lack a consensus motif
E2F might bind core promoter region that lacks a consensus E2F motif by looping from a consensus motif located in an upstream or downstream enhancer.  E2F1 would bind a distal consensus motif and engage in protein-protein interactions wit a factor bound in core promoter region.

To rule out that recruitment of E2F1 to core promoters that lack a consensus is via looping from consensus sites, remove promoter from its normal chromosomal environment and determine whether E2F1 can continue to bind isolated region.

Use stably transmitted, autonomously replicating nuclear extrachromosomal episomal vectors.

Create stable cell lines containing episomes harbouring promoters with a consensus E2F site with high enrichment values in E2F ChIP-chip assas.  Analyse binding to episomes by using primers specific to regions of episomal vector flanking cloning site. Episomes harbouring MYC, CDC23 and HIST1H3F promoters were all enriched in E2F1 and E2F4 ChIP samples but not in IgG samples.

Create stable cells containing episomes harbouring promoters with a consensus E2f site but did not show high enrichment values in E2F ChIP-chip assays. Not bound by E2F1 or E2F4 in eChIP assays though they contain a consensus E2F site.

HIST1HI1D core promoter regions which contain consensus E2F motifs and show high enrichment in ChIP-chip assays are bound by Ef2s when analysed in eChIP assays.

Select 8 promoter without consensus E2F motif, clone promoter fragments into episomal vector and create stable cell lines containing each construct. Perform eChIP assays using E2F antibodies and analyse binding to episomes and endogenous MYC promoter. 7 out of 9 E2F target promoters that lacked a consensus E2F site were bound by E2F1 when isolated from normal chromatin contexdt and analysed in eChIP assay.

Does not suport model that E2F is detected at core promoter due to protein-protein interactions mediated by looping from a distal consensus motif.
Establishment of the eChIP assay. (Left) Analysis of E2F1 and E2F4 ChIP samples in cells harboring the episomal negative controls: the empty episomal vector (left, bottom) and an episome containing a portion of the transcribed region of CRAMP1L (lefttop). (Middle) Binding of E2F1 and E2F4 to episomes containing a 500-bp fragment of the MYC, CDC23, or HIST1H3F promoters, all of which possess a consensus E2F motif and were previously shown to be bound by E2Fs in the ChIP-chip experiments. (Right) Binding of E2F1 and E2F4 to episomes containing a 500-bp fragment of the RASSF5, RAG2, or PGBD3 promoters, all of which possess a consensus E2F motif, but were previously shown not to be bound by E2Fs in the ChIP-chip experiments. For all experiments, IgG was used as a negative control antibody and binding to the endogenous MYC promoter was analyzed as a positive control for E2F enrichment. Furthermore, the binding of E2F1 and E2F4 to the corresponding endogenous promoters was analyzed in all of the eChIP samples.

Mapping binding site using eChIP
A 3rd model that could account for E2F recruitment to regions that lack a consensus motif is asssisted binding. DNA-binding domain of E2F1 is necessary (as HA-DBD mut e2F1 cannot bind promoters) and E2Fs would bind sites that weakly match PWM but lack a consensus motif.

Although both target and nontarget promoters on array contain matches to PWM, binding of E2F1 to target promoters could be enhanced due to specific interactions of E2F1 with other factors that bind target but not to nontarget set of promoters.

Predicts that best match to E2F PWM in core promoter region is require for recruitment to E2F1. Test by inserting progressively smaller fragments into episomal vector.

Test for HIST1H1D and TIMELESS promoters which show high enrichment but do not contain E2F motifs in core promoter tregions. Compare binding of E2F1 and E2F4 to a 500bp and 150 bp region of HIST1H1D promoter. 150bp fragment has best mnatch to E2F PWM. It is sufficient for rescruitment of E2Fs in eChIP assay. Binding of E2Fs was tested for TIMELESS promoter in eChIP assay. 500bp and 300bp fragments could recruit E2F1 but not 150bp fragment.  Best match to E2F PWM is not sufficient for E2F1 binding.

To determine whether best match to E2F PWM is necessary to recruit E2F family members to TIMELESS promoter, create construct containing 300 nt of core promoter region but lacking E2F PWM. Create stable cell line with this episomal construct. Test binding. Deletion of E2F PWM eliminated binding of E2F1 and E2F4 to TIMELESS promoter.

Thus a region containing best match to E2F PWM is necessary but not sufficient to recruit E2F to TIMELESS promoter. Supports assisted binding model.

Discussion
Correlation in occupancy of E2F site and location relative to transcription start site. Consensus motifs not bound by E2F family members are usually not located in core promoter elements. 8 in 9 tested regions with E2F consensus motifs not bound by E2F4 did not have histone modifications noramally associated with core promoter regions. Many of these regions semed to be in actively transcribed region, suggetsing in MCF7 cells those genes must use different promoters.

Factors specifying whether E2F is recruited to an in vivo binding site
1. site must be in a core promoter
2. promoter must be used as a promoter by the transcriptional machinery in that particular cell type.



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