Accessibility of promoter DNA is not the primary determinant of chromatin-mediated gene regulation

DNA accessibility is thought to be of major importance in regulating gene expression. We test this hypothesis using a restriction enzyme as a probe of chromatin structure and as a proxy for transcription factors. We measured the digestion rate and the fraction of accessible DNA at almost all genomic AluI sites in budding yeast and mouse liver nuclei. Hepatocyte DNA is more accessible than yeast DNA, consistent with longer linkers between nucleosomes, and suggesting that nucleosome spacing is a major determinant of accessibility. DNA accessibility varies from cell to cell, such that essentially no sites are accessible or inaccessible in every cell. AluI sites in inactive mouse promoters are accessible in some cells, implying that transcription factors could bind without activating the gene. Euchromatin and heterochromatin have very similar accessibilities, suggesting that transcription factors can penetrate heterochromatin. Thus, DNA accessibility is not likely to be the primary determinant of gene regulation.Fil: Chereji, Razvan V.. National Institutes of Health; Estados UnidosFil: Eriksson, Peter R.. National Institutes of Health; Estados UnidosFil: Ocampo, Josefina. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Prajapati, Hemant Kumar. National Institutes of Health; Estados UnidosFil: Clark, David. National Institutes of Health; Estados Unido

Contrasting roles of the RSC and ISW1/CHD1 chromatin remodelers in RNA polymerase II elongation and termination

Most yeast genes have a nucleosome-depleted region (NDR) at the promoter and an array of regularly spaced nucleosomes phased relative to the transcription start site. We have examined the interplay between RSC (a conserved essential SWI/SNF-type complex that determines NDR size) and the ISW1, CHD1, and ISW2 nucleosome spacing enzymes in chromatin organization and transcription, using isogenic strains lacking all combinations of these enzymes. The contributions of these remodelers to chromatin organization are largely combinatorial, distinct, and nonredundant, supporting a model in which the +1 nucleosome is positioned by RSC and then used as a reference nucleosome by the spacing enzymes. Defective chromatin organization correlates with altered RNA polymerase II (Pol II) distribution. RSC-depleted cells exhibit low levels of elongating Pol II and high levels of terminating Pol II, consistent with defects in both termination and initiation, suggesting that RSC facilitates both. Cells lacking both ISW1 and CHD1 show the opposite Pol II distribution, suggesting elongation and termination defects. These cells have extremely disrupted chromatin, with high levels of closely packed dinucleosomes involving the second (+2) nucleosome. We propose that ISW1 and CHD1 facilitate Pol II elongation by separating closely packed nucleosomes.Fil: Ocampo, Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Chereji, Razvan V.. National Institutes of Health; Estados UnidosFil: Eriksson, Peter R.. National Institutes of Health; Estados UnidosFil: Clark, David. National Institutes of Health; Estados Unido

Noise and interlocking signaling pathways promote distinct transcription factor dynamics in response to different stresses

All cells perceive and respond to environmental stresses through elaborate stress-sensing networks. Yeast cells sense stress through diverse signaling pathways that converge on the transcription factors Msn2 and Msn4, which respond by initiating rapid, idiosyncratic cycles into and out of the nucleus. To understand the role of Msn2/4 nuclear localization dynamics, we combined time-lapse studies of Msn2-GFP localization in living cells with computational modeling of stress-sensing signaling networks. We find that several signaling pathways, including Ras/protein kinase A, AMP-activated kinase, the high-osmolarity response mitogen-activated protein kinase pathway, and protein phosphatase 1, regulate activation of Msn2 in distinct ways in response to different stresses. Moreover, we find that bursts of nuclear localization elicit a more robust transcriptional response than does sustained nuclear localization. Using stochastic modeling, we reproduce in silico the responses of Msn2 to different stresses, and demonstrate that bursts of localization arise from noise in the signaling pathways amplified by the small number of Msn2 molecules in the cell. This noise imparts diverse behaviors to genetically identical cells, allowing cell populations to “hedge their bets” in responding to an uncertain future, and to balance growth and survival in an unpredictable environment

Self‐report versus clinician‐ratings in the assessment of aggression in violent offenders

Background: The construct of aggression is central to work with violent offenders, but it is a broad construct that can be assessed by many different methods and instruments. Its measurement may, however, have profound implications for treatment planning. We need more knowledge about how different methods for assessing aggression relate to each other. Aims: Our aims were to investigate, first, the convergence and concordance of two methods of assessing aggression: self-report and clinical assessment and, second, to determine the degree to which aggression can be discriminated from neighbouring constructs, such as hostility, anger and criminal behaviour. Methods: A nationally representative Swedish cohort of 269 18–25-year-old incarcerated violent offenders was recruited. Data were collected through structured self-reports of aggression, anger and hostility traits (Aggression Questionnaire-Revised Swedish Version) and clinical assessments of lifetime prevalence of aggressive and antisocial behaviours (Life History of Aggression). Criminal records were retrieved from the Swedish National Crime Register. Results: Self-ratings and clinician-ratings of aggression were highly convergent and concordant, especially regarding physical aggression. Violent offence records were weakly, if at all, correlated, while self-reported hostility was weakly, or not at all, correlated with self-reported or with clinician-rated aggression. There was an inverse relationship between aggression and criminal records of sexual offences. Conclusions and Implications: Even though a combination of self-reports and clinician-ratings may provide a better overview of an individual's aggressive behaviours, our results indicate that they yield such similar information that either alone would be sensitive enough. Our results do not, however, support using one of these methods as a proxy for the other since choice of measure and accepted concordance between them depend on the context within which the assessment is conducted. We reconfirmed that official records of violent offending are unlikely to be adequate measures of outcome after interventions to reduce aggressive behaviours

Mediator binds to boundaries of chromosomal interaction domains and to proteins involved in DNA looping, RNA metabolism, chromatin remodeling, and actin assembly

Mediator is a multi-unit molecular complex that plays a key role in transferring signals from transcriptional regulators to RNA polymerase II in eukaryotes. We have combined biochemical purification of the Sac-charomyces cerevisiae Mediator from chromatin with chromatin immunoprecipitation in order to reveal Mediator occupancy on DNA genome-wide, and to identify proteins interacting specifically with Mediator on the chromatin template. Tandem mass spectrometry of proteins in immunoprecipitates of mediator complexes revealed specific interactions between Mediator and the RSC, Arp2/Arp3, CPF, CF 1A and Lsm complexes in chromatin. These factors are primarily involved in chromatin remodeling, actin assembly, mRNA 3'-end processing, gene looping and mRNA decay, but they have also been shown to enter the nucleus and participate in Pol II transcription. Moreover, we have found that Mediator, in addition to binding Pol II promoters, occupies chromosomal interacting domain (CID) boundaries and that Mediator in chromatin associates with proteins that have been shown to interact with CID boundaries, such as Sth1, Ssu72 and histone H4. This suggests that Mediator plays a significant role in higher-order genome organization