Ian B. Dodd

Theoretical Analysis of Epigenetic Cell Memory by Nucleosome Modification

Chromosomal regions can adopt stable and heritable alternative states resulting in bistable gene expression without changes to the DNA sequence. Such epigenetic control is often associated with alternative covalent modifications of histones. The stability and heritability of the states are thought to involve positive feedback where modified nucleosomes recruit enzymes that similarly modify nearby nucleosomes. We developed a simplified stochastic model for dynamic nucleosome modification based on the silent mating-type region of the yeast Schizosaccharomyces pombe. We show that the mechanism can give strong bistability that is resistant both to high noise due to random gain or loss of nucleosome modifications and to random partitioning upon DNA replication. However, robust bistability required: (1) cooperativity, the activity of more than one modified nucleosome, in the modification reactions and (2) that nucleosomes occasionally stimulate modification beyond their neighbor nucleosomes, arguing against a simple continuous spreading of nucleosome modification.

Systematic method for the detection of potential λ Cro-like DNA-binding regions in proteins

We have developed and tested a systematic method for the location and statistical evaluation of potential DNA-binding regions of the lambda Cro type in protein sequences. Using this approach to examine proteins expected to contain such regions, we have been able to compile a statistically homogeneous master set of 37 lambda Cro-like DNA-binding domains. Examination of a protein database revealed other prokaryotic proteins that are similar to this lambda Cro-like group. There are also many DNA-binding proteins that are not found to be significantly similar to the lambda Cro group, consistent with previous suggestions that different types of protein sequence may be able to achieve a similar mode of binding and that there exist other modes of sequence-specific DNA-binding. A useful feature of the method is that it can be applied without a computer.

Potent transcriptional interference by pausing of RNA polymerases over a downstream promoter

Elongating RNA polymerases (RNAPs) can interfere with transcription from downstream promoters by inhibiting DNA binding by RNAP and activators. However, combining quantitative measurement with mathematical modeling, we show that simple RNAP elongation cannot produce the strong asymmetric interference observed between a natural face-to-face promoter pair in bacteriophage lambda. Pausing of elongating polymerases over the RNAP-binding site of the downstream promoter is demonstrated in vivo and is shown by modeling to account for the increased interference. The model successfully predicts the effects on interference of treatments increasing or reducing pausing. Gene regulation by pausing-enhanced occlusion provides a general and potentially widespread mechanism by which even weak converging or tandem transcription, either coding or noncoding, can bring about strong in cis repression.

Ultrasensitive gene regulation by positive feedback loops in nucleosome modification

Eukaryotic transcription involves the synergistic interaction of many different proteins. However, the question remains how eukaryotic promoters achieve ultrasensitive or threshold responses to changes in the concentration or activity of a single transcription factor (TF). We show theoretically that by recruiting a histone‐modifying enzyme, a TF binding non‐cooperatively to a single site can change the balance between opposing positive feedback loops in histone modification to produce a large change in gene expression in response to a small change in concentration of the TF. This mechanism can also generate bistable promoter responses, allowing a gene to be on in some cells and off in others, despite the cells being in identical conditions. In addition, the system provides a simple means by which the activities of many TFs could be integrated at a promoter.

Control of gene expression in the P2-related template coliphages. III: DNA sequence of the major control region of phage 186

The PstI fragment (65.5% to 77.4%) of coliphage 186, known genetically to encode the major control genes, has been sequenced, and an analysis performed to assess coding capacity, transcription-translation signals, and to identify any other significant features. Our analysis indicates that the region encodes: seven genes, including the int and cI genes, which overlap, the late control gene B, and two genes, named CP75 and CP76, encoding potential DNA-binding proteins; a promoter pB and terminator tB for the rightward transcription of the B gene, and we predict the existence of this transcript in a lysogen; a promoter pL and terminator tL for leftward transcription that encodes the int and cI genes, and represents the presumed lysogenic transcript; a promoter pR for rightward transcription to give the presumed (early) lytic transcript that is overlapping and convergent with the lysogenic transcript; and finally, a potential operator site for repressor binding in the region of the pR promoter. Preliminary evidence is presented to support this analysis.

Structure‐activity relationships within the N‐terminal short consensus repeats (SCR) of human CR1 (C3b / C4b receptor, CD35): SCR 3 plays a critical role in inhibition of the classical and alternative pathways of complement activation

Genes coding for between one and four short consensus repeats (SCR) of the N‐terminal region of human complement receptor 1 (CR1) were synthesized from oligonucleotides and those encoding SCR(1 – 2), SCR(1 – 3), SCR(1 – 4), SCR3 and SCR(3 – 4) were expressed as inclusion bodies in Escherichia coli. Following solubilization in urea, the proteins were partially purified and refolded and the activity of each protein was assessed in both classical and alternative pathway complement assays. All fragments showed a varying degree of activity with the general order being SCR(1 – 3) ≡ SCR(1 – 4) > SCR(1 – 2). Addition of SCR3 to SCR(1 – 2) significantly improved potency, whereas the addition of SCR4 conferred no additional benefit. This observation, coupled with the ability of the single‐domain SCR3 to inhibit classical pathway mediated lysis with an IH50% (inhibition of hemolysis by 50 %) of 4.8 μM, demonstrates that SCR3 provides key binding interactions with activated complement components. SCR(1 – 3) was able to inhibit both classical and alternative pathways of complement activation, showing that the N‐terminal SCR of CR1 retain the ability to interact with C3b. Assays for CR1‐like cofactor activity for factor I using C4b‐like C4 or C3b‐like C3 as substrates showed that SCR(1 – 3) possessed such cofactor activity and that C4b‐like C4 was a better substrate. When compared to full‐length (30 SCR) soluble CR1 (sCR1), SCR(1 – 3) was significantly less potent in accord with a model involving multi‐valent binding of C3b / C4b to CR1.

Identification of residues in the N-terminal PAS domains important for dimerization of Arnt and AhR

The basic helix–loop–helix (bHLH).PAS dimeric transcription factors have crucial roles in development, stress response, oxygen homeostasis and neurogenesis. Their target gene specificity depends in part on partner protein choices, where dimerization with common partner Aryl hydrocarbon receptor nuclear translocator (Arnt) is an essential step towards forming active, DNA binding complexes. Using a new bacterial two-hybrid system that selects for loss of protein interactions, we have identified 22 amino acids in the N-terminal PAS domain of Arnt that are involved in heterodimerization with aryl hydrocarbon receptor (AhR). Of these, Arnt E163 and Arnt S190 were selective for the AhR/Arnt interaction, since mutations at these positions had little effect on Arnt dimerization with other bHLH.PAS partners, while substitution of Arnt D217 affected the interaction with both AhR and hypoxia inducible factor-1α but not with single minded 1 and 2 or neuronal PAS4. Arnt uses the same face of the N-terminal PAS domain for homo- and heterodimerization and mutational analysis of AhR demonstrated that the equivalent region is used by AhR when dimerizing with Arnt. These interfaces differ from the PAS β-scaffold surfaces used for dimerization between the C-terminal PAS domains of hypoxia inducible factor-2α and Arnt, commonly used for PAS domain interactions.

Action at a distance in CI repressor regulation of the bacteriophage 186 genetic switch.

The non‐lambdoid coliphage 186 provides an alternative model to the lytic‐lysogenic switch of phage λ. Like λ, the key switch regulator, the CI repressor, associates to octamers. Unlike λ, the lytic promoter (pR) and the lysogenic promoter (pL) are face‐to‐face, 62 bp apart and are flanked by distal CI binding sites (FL and FR) located ≈ 300 bp away. Using reporter and footprinting studies, we show that the outcome, but not the mechanism, of regulation by 186 CI is very similar to λ. 186 CI stimulates pL transcription indirectly by repressing convergent interfering transcription from pR. However, in the absence of the flanking FL and FR sites, CI bound at pR interacts co‐operatively with a weak CI binding site at pL and represses both promoters. FL and FR play a critical role; they assist repression of pR and simultaneously alleviate repression of pL, thus allowing high pL activity. We propose that the 186 switch is regulated by a novel mechanism in which a CI octamer bound at pR forms alternative DNA loops to pL or to a flanking site, depending on CI concentration.

Theory for the stability and regulation of epigenetic landscapes.

Cells can often choose among several stably heritable phenotypes. Examples are the expressions of genes in eukaryotic cells where long chromosomal regions can adopt persistent and heritable silenced or active states that may be associated with positive feedback in dynamic modification of nucleosomes. We generalize this mechanism in terms of bistability associated with valleys in an epigenetic landscape. A transfer matrix method was used to rigorously follow the system through the disruptive process of cell division. This combined treatment of noisy dynamics both between and during cell division provides an efficient way to calculate the stability of alternative states in a broad range of epigenetic systems.

The generation of promoter-mediated transcriptional noise in bacteria.

Noise in the expression of a gene produces fluctuations in the concentration of the gene product. These fluctuations can interfere with optimal function or can be exploited to generate beneficial diversity between cells; gene expression noise is therefore expected to be subject to evolutionary pressure. Shifts between modes of high and low rates of transcription initiation at a promoter appear to contribute to this noise both in eukaryotes and prokaryotes. However, models invoked for eukaryotic promoter noise such as stable activation scaffolds or persistent nucleosome alterations seem unlikely to apply to prokaryotic promoters. We consider the relative importance of the steps required for transcription initiation. The 3-step transcription initiation model of McClure is extended into a mathematical model that can be used to predict consequences of additional promoter properties. We show in principle that the transcriptional bursting observed at an E. coli promoter by Golding et al. (2005) can be explained by stimulation of initiation by the negative supercoiling behind a transcribing RNA polymerase (RNAP) or by the formation of moribund or dead-end RNAP-promoter complexes. Both mechanisms are tunable by the alteration of promoter kinetics and therefore allow the optimization of promoter mediated noise.