Ryan A. Irvine

Multiple signal input and output domains of the 160-kilodalton nuclear receptor coactivator proteins.

ABSTRACT Members of the 160-kDa nuclear receptor coactivator family (p160 coactivators) bind to the conserved AF-2 activation function found in the hormone binding domains of nuclear receptors (NR) and are potent transcriptional coactivators for NRs. Here we report that the C-terminal region of p160 coactivators glucocorticoid receptor interacting protein 1 (GRIP1), steroid receptor coactivator 1 (SRC-1a), and SRC-1e binds the N-terminal AF-1 activation function of the androgen receptor (AR), and p160 coactivators can thereby enhance transcriptional activation by AR. While they all interact efficiently with AR AF-1, these same coactivators have vastly different binding strengths with and coactivator effects on AR AF-2. p160 activation domain AD1, which binds secondary coactivators CREB binding protein (CBP) and p300, was previously implicated as the principal domain for transmitting the activating signal to the transcription machinery. We identified a new highly conserved motif in the AD1 region which is important for CBP/p300 binding. Deletion of AD1 only partially reduced p160 coactivator function, due to signaling through AD2, another activation domain located at the C-terminal end of p160 coactivators. C-terminal coactivator fragments lacking AD1 but containing AD2 and the AR AF-1 binding site served as efficient coactivators for full-length AR and AR AF-1. The two signal input domains (one that binds NR AF-2 domains and one that binds AF-1 domains of some but not all NRs) and the two signal output domains (AD1 and AD2) of p160 coactivators played different relative roles for two different NRs: AR and thyroid hormone receptor.

Contribution of the Androgen Receptor to Prostate Cancer Predisposition and Progression

Although prostate cancer is heterogeneous in its etiology and progression, androgen signaling through the androgen receptor (AR) appears to be involved in all aspects of the disease, from initiation to development of treatment resistance. Lifetime exposure to a constitutively more active AR, encoded by AR alleles as defined by two translated polymorphic microsatellites (CAG and GGC), results in a significant increase in prostate cancer risk. The AR gene is amplified or a target for somatic gain-of-function mutations in metastatic prostate cancer. Gain-of-function AR gene mutations may result in inappropriate activation of the AR, thereby contributing to the failure of conventional androgen-ablation treatments. In cases where no genetically altered receptors are observed, altered signaling through the AR, achieved by cross-talk with other signaling pathways (e.g. kinase-mediated pathways) and/or inappropriate expression of coregulatory proteins, may contribute to disease progression. Thus, the AR-signaling axis contributes to many aspects of prostate cancer, including initiation, progression and resistance to current forms of therapy. This recognition represents a paradigm shift in our understanding of the molecular mechanisms involved in progression of prostate cancer, and provides insight into novel AR-targeted therapies which ultimately may be more effective than current forms of androgen ablation.

DNA methylation has a local effect on transcription and histone acetylation.

ABSTRACT DNA methylation is commonly associated with gene silencing, and a link between histone deacetylation and DNA methylation has been established. However, the transcriptional impact of the position and length of methylated zones relative to the promoter and the coding region of a gene remains quite unclear. This study investigates the impact of regional methylation on transcription and the relationship between DNA methylation and histone acetylation. Using patch-methylated stable episomes in human cells, we establish the pivotal importance of the location of DNA methylation in the regulation of transcription. We further demonstrate that the size of the methylated patch is not a key determinant for transcriptional suppression. The impact of DNA methylation on transcription is greater when it is in the transcription unit, and it is primarily a local effect. However, methylation outside of the transcription unit may potentiate the effect of methylation within the transcription unit. Acetylated histones are associated with unmethylated DNA and are nearly absent from methylated DNA regions. This association appears to be local and does not propagate along the DNA.

Generation and characterization of endonuclease G null mice.

ABSTRACT Endonuclease G (endo G) is one of the most abundant nucleases in eukaryotic cells. It is encoded in the nucleus and imported to the mitochondrial intermembrane space. This nuclease is active on single- and double-stranded DNA. We genetically disrupted the endo G gene in mice without disturbing a conserved, overlapping gene of unknown function that is oriented tail to tail with the endo G gene. In these mice, the production of endo G protein is not detected, and the disruption abolishes the nuclease activity of endo G. The absence of endo G has no effect on mitochondrial DNA copy number, structure, or mutation rate over the first five generations. There is also no obvious effect on nuclear DNA degradation in standard apoptosis assays. The endo G null mice are viable and show no age-related or generational abnormalities anatomically or histologically. We infer that this highly conserved protein has no mitochondrial or apoptosis function that can discerned by the assays described here and that it may have a function yet to be determined. The early embryonic lethality of endo G null mice recently reported by others may be due to the disruption of the gene that overlaps the endo G gene.

The embryonic lethality in DNA ligase IV-deficient mice is rescued by deletion of Ku: implications for unifying the heterogeneous phenotypes of NHEJ mutants

There are two general pathways by which multicellular eukaryotes repair double-strand DNA breaks (DSB): homologous recombination (HR) and nonhomologous DNA end joining (NHEJ). All mammalian mutants in the NHEJ pathway demonstrate a lack of B and T lymphocytes and ionizing radiation sensitivity. Among these NHEJ mutants, the DNA-PK(cs) and Artemis mutants are the least severe, having no obvious phenotype other than the general defects described above. Ku mutants have an intermediate severity with accelerated senescence. The XRCC4 and DNA ligase IV mutants are the most severe, resulting in embryonic lethality. Here we show that the lethality of DNA ligase IV-deficiency in the mouse can be rescued when Ku86 is also absent. To explain the fact that simultaneous gene mutations in the NHEJ pathway can lead to viability when a single mutant is not viable, we propose a nuclease/ligase model. In this model, disrupted NHEJ is more severe if the Artemis:DNA-PK(cs) nuclease is present in the absence of a ligase, and Ku mutants are of intermediate severity, because the nuclease is less efficient. This model is also consistent with the order of severity in organismal phenotypes; consistent with chromosomal breakage observations reported here; and consistent with the NHEJ mutation identified in radiation sensitive human SCID patients.