Amyloid Burden in Alzheimer's Disease Patients Is Associated with Alterations in Circadian Rhythm.

BACKGROUND AND PURPOSE: In this study we evaluated the relationship between amyloid-beta (Aß) deposition and 3 aspects of sleep quality in a group of clinically diagnosed Alzheimer's disease (AD) patients. METHODS: We used self-report questionnaires to assess the quality of sleep using 3 previously established surveys: the Glasgow Sleep Effort Scale (GSES), the Pittsburgh Sleep Quality Index (PSQI), and the Morningness-Eveningness Questionnaire (MEQ). These questionnaires focused on the sleep effort, sleep efficiency, and circadian rhythm patterns of each participant. Also, we evaluated the regional distribution of Aß in the brain by amyloid positron emission tomography-computed tomography (PET-CT) standardized uptake value ratios (SUVRs) in healthy normal (HN), mild cognitive impairment (MCI), and AD dementia groups. The MCI and AD dementia groups were combined to form the group with cognitive impairment due to AD (CIAD). RESULTS: GSES and MEQ scores differed significantly between the HN, MCI, and AD dementia groups (p<0.037), whereas PSQI scores were similar across the groups (p=0.129). GSES and MEQ scores also differed between the HN and CIAD groups (p<0.018). Circadian rhythm scores positively correlated with amyloid PET-CT SUVR in posterior cingulate cortices (p<0.049). CONCLUSIONS: Sleep effort and abnormal shifts in circadian rhythm were more significant in the CIAD group than in the HN group. At the same time, HN subjects had minimal sleep disturbance, irrespective of clinical status. Thus, alterations in circadian rhythm may be indicative of neurodegeneration due to Aß deposition.

Genomic analyses of transcription factor binding, histone acetylation, and gene expression reveal mechanistically distinct classes of estrogen-regulated promoters.

To explore the global mechanisms of estrogen-regulated transcription, we used chromatin immunoprecipitation coupled with DNA microarrays to determine the localization of RNA polymerase II (Pol II), estrogen receptor alpha (ERalpha), steroid receptor coactivator proteins (SRC), and acetylated histones H3/H4 (AcH) at estrogen-regulated promoters in MCF-7 cells with or without estradiol (E2) treatment. In addition, we correlated factor occupancy with gene expression and the presence of transcription factor binding elements. Using this integrative approach, we defined a set of 58 direct E2 target genes based on E2-regulated Pol II occupancy and classified their promoters based on factor binding, histone modification, and transcriptional output. Many of these direct E2 target genes exhibit interesting modes of regulation and biological activities, some of which may be relevant to the onset and proliferation of breast cancers. Our studies indicate that about one-third of these direct E2 target genes contain promoter-proximal ERalpha-binding sites, which is considerably more than previous estimates. Some of these genes represent possible novel targets for regulation through the ERalpha/AP-1 tethering pathway. Our studies have also revealed several previously uncharacterized global features of E2-regulated gene expression, including strong positive correlations between Pol II occupancy and AcH levels, as well as between the E2-dependent recruitment of ERalpha and SRC at the promoters of E2-stimulated genes. Furthermore, our studies have revealed new mechanistic insights into E2-regulated gene expression, including the absence of SRC binding at E2-repressed genes and the presence of constitutively bound, promoter-proximally paused Pol IIs at some E2-regulated promoters. These mechanistic insights are likely to be relevant for understanding gene regulation by a wide variety of nuclear receptors.

Folate-Dependent Cognitive Impairment Associated With Specific Gene Networks in the Adult Mouse Hippocampus.

Short-term folate deficiency has been linked to cognitive defects. Given folate's role in regulating nucleotide synthesis and DNA and histone methylation, these changes are often linked to altered gene expression and might be controlled by specific regulatory networks. In our study we examined the effects of folic acid (FA) deficient or replete diets in mice, containing either no source of folate or normal FA intake, beginning post-weaning and persisting through the end of adult life at 18 months. Our goal was to assess levels of cognition in these mice using the novel object test and then connect the cognitive results to genetic changes. FA deficient mice showed significant memory impairment compared to control counterparts beginning at 5 months and persisting through 17 months, as determined by the novel object test. These deficits were associated with 363 significantly downregulated and 101 significantly upregulated genes in the deficient condition compared to the control condition in microarray analysis of hippocampal tissue. Many of these gene expression changes were determined to be specific to the hippocampus. Significant ontological categories for differential genes included nucleotide regulation, ion channel activity, and MAPK signaling; while some of these categories contain genes previously mapped to cognitive decline, other genes have not previously been associated with cognition. To determine proteins possibly involved in regulation of these genes, we performed bioinformatics analysis and found enriched motifs of for MafB and Zfp410 binding sites. These genes and enriched motifs may represent targets for treatment or investigation of memory-related diseases.

High Performance Liquid Chromatography Separation of Epigenetic Cytosine Variants.

Epigenetic modifications enable cells to genetically respond to chemical inputs from environmental sources. These marks play a pivotal role in normal biological processes (e.g., differentiation, host defense and metabolic programs) but also contribute to the development of a wide variety of pathological conditions (e.g., cancer and Alzheimer's disease). In particular, DNA methylation represents very stable epigenetic modification of cytosine bases that is strongly associated with a reduction in gene activity. Although High Performance Liquid Chromatography (HPLC) methodologies have been used to resolve methylated cytosine from unmodified cytosine bases, these represent only two of the five major cytosine analogs in the cell. Moreover, failure to resolve these other cytosine analogs might affect an accurate description of the cytosine methylation status in cells. In this present study, we determined the HPLC conditions required to separate the five cytosine analogs of the methylation/demethylation pathway. This methodology not only provides a means to analyze cytosine methylation as a whole, but it could also be used to more accurately calculate the methylation ratio from biological samples.

Amyloid-ß alters the DNA methylation status of cell-fate genes in an Alzheimer's disease model.

Alzheimer's disease (AD) is characterized by neurofibrillary tangles and extracellular amyloid-ß plaques (Aß). Despite ongoing research, some ambiguity remains surrounding the role of Aß in the pathogenesis of this neurodegenerative disease. While several studies have focused on the mutations associated with AD, our understanding of the epigenetic contributions to the disease remains less clear. To that end, we determined the changes in DNA methylation in differentiated human neurons with and without Aß treatment. DNA was isolated from neurons treated with Aß or vehicle, and the two samples were digested with either a methylation-sensitive (HpaII) or a methylation-insensitive (MspI) restriction endonuclease. The fragments were amplified and co-hybridized to a commercial promoter microarray. Data analysis revealed a subset of genomic loci that shows a significant change in DNA methylation following Aß treatment in comparison to the control group. After mapping these loci to nearby genes, we discovered high enrichment for cell-fate genes that control apoptosis and neuronal differentiation. Finally, we incorporated three of those genes in a possible model suggesting the means by which Aß contributes to the brain shrinkage and memory loss seen in AD.

Estrogen regulates JNK1 genomic localization to control gene expression and cell growth in breast cancer cells.

Steroid hormone and MAPK signaling pathways functionally intersect, but the molecular mechanisms of this cross talk are unclear. Here, we demonstrate a functional convergence of the estrogen and c-Jun N-terminal kinase 1 (JNK1) signaling pathways at the genomic level in breast cancer cells. We find that JNK1 binds to many promoters across the genome. Although most of the JNK1-binding sites are constitutive, a subset is estrogen regulated (either induced on inhibited). At the estrogen-induced sites, estrogen receptor (ER)α is required for the binding of JNK1 by promoting its recruitment to estrogen response elements or other classes of DNA elements through a tethering mechanism, which in some cases involves activating protein-1. At estrogen-regulated promoters, JNK1 functions as a transcriptional coregulator of ERα in a manner that is dependent on its kinase activity. The convergence of ERα and JNK1 at target gene promoters regulates estrogen-dependent gene expression outcomes, as well as downstream estrogen-dependent cell growth responses. Analysis of existing gene expression profiles from breast cancer biopsies suggests a role for functional interplay between ERα and JNK1 in the progression and clinical outcome of breast cancers.

Multiple sequence-specific DNA-binding proteins mediate estrogen receptor signaling through a tethering pathway.

The indirect recruitment (tethering) of estrogen receptors (ERs) to DNA through other DNA-bound transcription factors (e.g. activator protein 1) is an important component of estrogen-signaling pathways, but our understanding of the mechanisms of ligand-dependent activation in this pathway is limited. Using proteomic, genomic, and gene-specific analyses, we demonstrate that a large repertoire of DNA-binding transcription factors contribute to estrogen signaling through the tethering pathway. In addition, we define a set of endogenous genes for which ERα tethering through activator protein 1 (e.g. c-Fos) and cAMP response element-binding protein family members mediates estrogen responsiveness. Finally, we show that functional interplay between c-Fos and cAMP response element-binding protein 1 contributes to estrogen-dependent regulation through the tethering pathway. Based on our results, we conclude that ERα recruitment in the tethering pathway is dependent on the ligand-induced formation of transcription factor complexes that involves interplay between the transcription factors from different protein families.

Postrecruitment regulation of RNA polymerase II directs rapid signaling responses at the promoters of estrogen target genes.

Under classical models for signal-dependent transcription in eukaryotes, DNA-binding activator proteins regulate the recruitment of RNA polymerase II (Pol II) to a set of target promoters. However, recent studies, as well as our results herein, show that Pol II is widely distributed (i.e., "preloaded") at the promoters of many genes prior to specific signaling events. How Pol II recruitment and Pol II preloading fit within a unified model of gene regulation is unclear. In addition, the mechanisms through which cellular signals activate preloaded Pol II across mammalian genomes remain largely unknown. We show here that the predominant genomic outcome of estrogen signaling is the postrecruitment regulation of Pol II activity at target gene promoters, likely through specific changes in Pol II phosphorylation rather than through recruitment of Pol II to the promoters. Furthermore, we show that negative elongation factor binds to estrogen target promoters in conjunction with preloaded Pol II and represses gene expression until the appropriate signal is received. Finally, our studies reveal that the estrogen-dependent activation of preloaded Pol II facilitates rapid gene regulatory responses which play important physiological roles in regulating estrogen signaling itself. Our results reveal a broad use of postrecruitment Pol II regulation by the estrogen signaling pathway, a mode of regulation that is likely to apply to a wide variety of signal-regulated pathways.