The Role of Epigenetic Regulation in Transcriptional Memory in the Immune System.

The immune system is exquisitely poised to identify, respond to, and eradicate pathogens from the body, as well as to produce a more rapid and augmented response to a subsequent encounter with the pathogen. These cellular responses rely on the highly coordinated and rapid activation of gene expression programs as well as the ability of the cell to retain a memory of the initial gene response. It is clear that chromatin structure and epigenetic mechanisms play a crucial role in determining these gene responses, and in fact the immune system has proved an instructive model for investigating the multifaceted mechanisms through which the chromatin landscape contributes to gene expression programs. These mechanisms include modifications to the DNA and histone proteins, the positioning, composition, and remodeling of nucleosomes, as well as the formation of higher-order chromatin structures. Moreover, it is now apparent that epigenetic mechanisms also provide an instrument by which cells can retain memory of the initial transcriptional response, "priming" the genome so that it can respond more quickly to subsequent exposure to the signal. Here, we use the immune system as a model to demonstrate the complex interplay between transcription factors and the chromatin landscape required to orchestrate precise gene responses to external stimuli and further to demonstrate how these interactions can establish memory of past transcriptional events. We focus on what we have learnt from the immune system and how this can inform our understanding of other cellular systems.

Reversing aberrant methylation patterns in cancer.

Changes to the epigenetic information within a cell play a significant role in cancer development and progression. These epigenetic changes are important in establishing the aberrant gene expression patterns that are a feature of cancer cell biology. We are currently experiencing a rapid advance in our understanding of how epigenetic information is written and interpreted in the cell, and the enzymes involved in these processes have been recognised as prime targets for therapeutic intervention. Reagents that target these enzymes have the potential to inhibit or reverse epigenetic changes in cancer cells. Evidence suggests that the aberrant regulation of two gene silencing pathways; involving DNA methylation and histone methylation, play an important role in cancer development. Considerable effort is being exerted in the development of inhibitors of these pathways. However, complex functional interactions exist between the DNA and histone methylation pathways, and these interactions will need to be considered in the design of inhibitory molecules. This review details current research into agents developed as inhibitors of these epigenetic pathways, focusing on the types of epigenetic modifications being targeted, interactions between these modifications and the use of these inhibitory agents in cancer treatment.

Determinants of a transcriptionally competent environment at the GM-CSF promoter.

Granulocyte macrophage-colony stimulating factor (GM-CSF) is produced by T cells, but not B cells, in response to immune signals. GM-CSF gene activation in response to T-cell stimulation requires remodelling of chromatin associated with the gene promoter, and these changes do not occur in B cells. While the CpG methylation status of the murine GM-CSF promoter shows no correlation with the ability of the gene to respond to activation, we find that the basal chromatin environment of the gene promoter influences its ability to respond to immune signals. In unstimulated T cells but not B cells, the GM-CSF promoter is selectively marked by enrichment of histone acetylation, and association of the chromatin-remodelling protein BRG1. BRG1 is removed from the promoter upon activation concomitant with histone depletion and BRG1 is required for efficient chromatin remodelling and transcription. Increasing histone acetylation at the promoter in T cells is paralleled by increased BRG1 recruitment, resulting in more rapid chromatin remodelling, and an associated increase in GM-CSF mRNA levels. Furthermore, increasing histone acetylation in B cells removes the block in chromatin remodelling and transcriptional activation of the GM-CSF gene. These data are consistent with a model in which histone hyperacetylation and BRG1 enrichment at the GM-CSF promoter, generate a chromatin environment competent to respond to immune signals resulting in gene activation.

Targeting epigenetic modifiers in cancer.

The chromatin structure of a gene plays an important role in regulating its expression. This structure is established through the action of various protein complexes that remodel nucleosomes, catalyse post-translational modifications, deposit histone variants and methylate DNA. Together these complexes establish epigenetic marks that influence expression of the gene. Some of these epigenetic marks are transient while others, such as those involved in silencing genes are more stable and can require several cell divisions to be fully implemented or reversed. Deregulated gene expression programs are a feature of cancer biology and it is now apparent that epigenetic changes, as well as genetic changes, are important in establishing these aberrant expression patterns. However, unlike genetic alterations, epigenetic changes are reversible. The complexes that catalyse these modifications therefore represent valuable targets for therapeutic intervention. Here we will review the most recent literature describing the protein complexes that catalyse epigenetic modifications and the inhibitors of these complexes that are being pursued as cancer drugs. In addition we will highlight those epigenetic modifiers that provide promise as therapeutic targets but for which inhibitors are not currently available.

Regulation of cytokine gene transcription in the immune system.

The controlled expression of cytokine genes is an essential component of an immune response. The specific types of cytokines as well as the time and place of their production is important in generating an appropriate immune response to an infectious agent. Aberrant expression is associated with pathological conditions of the immune system such as autoimmunity, atopy and chronic inflammation. Cytokine gene transcription is generally induced in a cell-specific manner. Over the last 15 years, a large amount of information has been generated describing the transcriptional controls that are exerted on cytokine genes. Recently, efforts have been directed at understanding how these genes are transcribed in a chromatin context. This review will discuss the mechanisms by which cytokine genes become available for transcription in a cell-restricted manner as well as the mechanisms by which these genes sense their environment and activate high level transcription in a transient manner. Particular attention will be paid to the role of chromatin in allowing transcription factor access to appropriate genes.

Functional interaction between the HIV transactivator Tat and the transcriptional coactivator PC4 in T cells.

The human immunodeficiency virus (HIV) transactivator Tat is a potent activator of transcription from the HIV long terminal repeat and is essential for efficient viral gene expression and replication. Tat has been shown to interact with components of the basal transcription machinery and transcriptional activators. Here we identify the cellular coactivator PC4 as a Tat-interacting protein using the yeast two-hybrid system and confirmed this interaction both in vitro and in vivo by coimmunoprecipitation. We found that this interaction has a functional outcome in that PC4 overexpression enhanced activation of the HIV long terminal repeat in transient transfection studies in a Tat-dependent manner. The domains of PC4 and Tat required for the interaction were mapped. In vitro binding studies showed that the basic transactivation-responsive binding domain of Tat is required for the interaction with PC4. The minimum region of PC4 required for Tat binding was amino acids 22-91, whereas mutation of the lysine-rich domain between amino acids 22 and 43 prevented interaction with Tat. Tat-PC4 interactions may be controlled by phosphorylation, because phosphorylation of PC4 by casein kinase II inhibited interactions with Tat both in vivo and in vitro. We propose that PC4 may be involved in linking Tat to the basal transcription machinery.

Localisation and expression of metallothionein immunoreactivity in the developing sheep brain.

Metallothioneins are small cysteine-rich proteins that bind heavy metals. In higher mammals there are complex families of metallothionein isoforms, which are well characterised at the DNA level but less so in terms of their cellular expression and function. In particular, little is known about the localisation of metallothionein in the developing mammalian brain. In this study using sheep fetuses, we have shown that metallothionein 1 and 2 isoform expression undergoes shifts in regional and cellular localisation during development of the brain. Metallothionein 1 and 2 expression is first detected by embryonic days E72 E73 (gestation is 150 days) at the mRNA level and the metallothionein protein is observed in cells of the proliferating ventricular zones. Subsequent expression is detected in radial glial cells, oligodendrocytes and astrocytes in several regions of the brain, most notably the cerebral cortex. In the adult brain, metallothionein is expressed in astrocytes but not in oligodendrocytes. Double-labelling immunohistochemistry using the glial fibrillary acidic protein (GFAP) an astrocyte marker, and metallothionein revealed that although there is an overlap in the profiles of the two proteins, there is no simple correlation in their expression. These observations are consistent with metallothionein, under physiological conditions, being regulated mainly by intracellular factors.

Metallothioneins 1 and 2 are expressed in the olfactory mucosa of mice in untreated animals and during the regeneration of the epithelial layer.

We have examined the expression of the MT1 and MT2 isoforms of metallothionein in the mouse olfactory mucosa. In untreated mice, metallothionein was strongly expressed in supporting cells, acinar cells of the Bowman's glands, and olfactory neurons. Expression was however restricted to a subset of cells within each type, and to zones within the olfactory system. Irrigation with ZnSO4 solution caused exfoliation of the olfactory epithelium and during the resultant regeneration, metallothionein immunoreactivity was associated with the proliferating basal cells. The ability to express MTs 1 and 2 did not appear to be obligatory for the early stages of regeneration since mice which do not express these isoforms responded similarly to wild type mice. Strong nuclear expression of metallothionein was noted in the untreated olfactory chamber following unilateral irrigation.

Human metallothionein gene MT1L mRNA is present in several human tissues but is unlikely to produce a metallothionein protein.

A human MT gene from the functional locus on chromosome 16, MT1L, is characterised and shown to produce mRNA in at least four human tissues. This gene is unlikely to produce a metallothionein protein because it contains a termination codon at position 26, by analogy to other human MT1 genes. MT1L cDNA is almost identical to another metallothionein cDNA clone reported recently, MT1R, suggesting that either there are unmapped human metallothionein genes, or that MT1L is polymorphic.

The spectrum and angular distribution of x rays scattered from a water phantom.

To calculate the response of an image receptor to the x rays emerging from a scattering medium, it is necessary to know the x-ray spectrum and intensity as a function of the angle of incidence on the receptor. To permit this calculation for any x-ray spectrum incident on a medium, these functions must be known for monoenergetic x rays. For monoenergetic x rays in the range 20-70 keV we have measured with a high-purity germanium detector the spectrum and intensity of x rays emitted from a water phantom at angles of 0 degree-50 degrees to the direction of the primary beam. The spectrum and intensity of emitted x rays have also been calculated by the Monte Carlo method. At small exit angles, most of the x rays have energies close to the incident energy. As the exit angle increases, the fraction of multiply scattered x rays increases. At very large exit angles, the dominant feature of the spectrum is the peak due to these multiply scattered x rays. For small scattering angles the Monte Carlo calculations are in good agreement with the measurements over the range of energies. For large scattering angeles the scattered photon fluence predicted by Monte Carlo modeling is consistently lower than the measurement in the region just below the full energy peak. The cause of the discrepancies is not fully understood, but cannot be accounted for by Compton broadening alone. An alternate approach to model incoherent scattering is proposed.

Characterisation of six additional human metallothionein genes.

Human metallothionein (MT) genes are clustered in a locus on chromosome 16, and this report presents the characterisation of the remaining six univestigated members of the family. Nucleotide sequencing in whole or part suggested that four of these genes, MT1I, MT1J, MT1K and MT1L do not encode expressed MT proteins, based on the presence of structural faults or atypical amino acid assignments. On the other hand, the structures of MT1H and MT1X are consistent with these genes being functional and encoding unique type 1 isoforms. The promoters of both genes conferred activity to CAT expression constructs when transfected into HeLa cells, and showed differential responses to inducers MT synthesis. Endogenous MT1H and MT1X genes were expressed at the mRNA level in HeLa cells following cadmium treatment. This work brings the number of functional class 1 and 2 MT genes in the human to eight, and confirms that each encodes structurally unique proteins.

Dose and quality control (DQC) in diagnostic radiology.

Dose and quality control in diagnostic radiology can play an important role in reducing x-ray exposure and costs whilst maintaining a high level of imaging quality and diagnostic benefit. It can also become very costly. Current government regulations demand unnecessary accuracy in the measurement and performance of certain parameters of x-ray generators whilst ignoring others which are more important. They totally neglect imaging systems. We urge a more critical approach to the requirements for dose and quality control programs. We propose the exchange of information through a user's club and a less regulatory but equally important role for government.

Speeds of film-screen combinations for radiography.

Values of Ex, the exposure required to yield a net optical density of 1.0, were determined for 12 blue- and nine green-sensitive film types used with 11 blue- and eight green-emitting screen types respectively. The measurements were made using x-rays generated at 80 kVp and 200 mA, under conditions simulating clinical procedures. To examine reciprocity law failure many of the determinations were repeated under similar conditions but at 15 mA. The values of Ex obtained at 15 mA were 6% to 93% greater than those at 200 mA.