Designer receptors for nucleotide-resolution analysis of genomic 5-Methylcytosine by cellular imaging

We report programmable receptors for the imaging-based analysis of 5-methylcytosine (5mC) in user-defined DNA sequences of single cells. Using fluorescent transcription-activator-like effectors (TALEs) that can recognize sequences of canonical and epigenetic nucleobases through selective repeats, we imaged cellular SATIII DNA, the origin of nuclear stress bodies (nSB). We achieve high nucleobase selectivity of natural repeats in imaging and demonstrate universal nucleobase binding by an engineered repeat. We use TALE pairs differing in only one such repeat in co-stains to detect 5mC in SATIII sequences with nucleotide resolution independently of differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a potential function of 5mC in its recruitment as initial step of nSB formation. This opens a new avenue for studying 5mC functions in chromatin regulation in situ with nucleotide, locus, and cell resolution

Alcoholism and Intimate Partner Violence: Effects on Children’s Psychosocial Adjustment

It is widely recognized that alcoholism and relationship violence often have serious consequences for adults; however, children living with alcoholic parents are susceptible to the deleterious familial environments these caregivers frequently create. Given the prevalence of IPV among patients entering substance abuse treatment, coupled with the negative familial consequences associated with these types of behavior, this review explores what have been, to this point, two divergent lines of research: (a) the effects of parental alcoholism on children, and (b) the effects of children’s exposure to intimate partner violence. In this article, the interrelationship between alcoholism and IPV is examined, with an emphasis on the developmental impact of these behaviors (individually and together) on children living in the home and offers recommendations for future research directions

Die transkriptionelle und epigenetische Rolle BRD4's bei der zellulären Stressantwort

Die zelluläre Stressantwort umfasst die Anpassung eines Organismus auf umweltbedingte und endogene Stressfaktoren, welche durch eine Vielzahl von molekularen Prozessen gesteuert wird. Eine Deregulierung dieser Antwort ist ein Indikator und möglicher Auslöser vieler Krankheiten, insbesondere Tumorerkrankungen, und bietet daher einen interessanten Angriffspunkt für Therapien. Ein sehr wichtiges und für die Tumorforschung auch aus therapeutischer Sicht äußerst vielversprechendes Protein ist das Bromodomänen enthaltende Protein 4, kurz BRD4. BRD4 spielt in sehr vielen Bereichen der Zelle eine wichtige Rolle, unter anderem als epigenetischer Sensor sowie transkriptioneller Regulator und ist damit ein wichtiges Bindeglied zwischen dem transkriptionellen Prozess und epigenetischen Mustern. Ziel dieser Arbeit war es, die Rolle von BRD4 bei der epigenetischen, als auch der transkriptionellen Regulation im Verlauf von zellulären Stressantworten zu untersuchen. Durch Genexpressionsanalysen in BRD4-defizienten Zellen, sowie Chromatin-ProteinInteraktionsstudien konnte ich 52 BRD4-regulierte Zielgene identifizieren, welche vor allem für Proteine der oxidativen Stressantwort sowie der Hitzestressantwort kodieren. Weiterführende Analysen identifizierten BRD4 als einen wichtigen Modulator eines der wichtigsten, mit oxidativem Stress assoziierten Signalwege, dem KEAP1/NRF2 Signalweg. Durch eine transkriptionelle Regulierung von KEAP1 kontrolliert BRD4 die Aktivität des Transkriptionsfaktors NRF2, welche wiederum die Expression zytoprotektiver Gene induziert. Eine Hemmung der BRD4 Aktivität führt unter Stress-Bedingungen zu einer Verringerung an reaktiven Sauerstoffspezies (ROS) in der Zelle und zu einem Schutz der Zellen vor oxidativem Stress-vermittelten Zelltod. Zudem konnte ich anhand einer Vielzahl von molekularbiologischen Experimenten zeigen, dass BRD4 direkt die Expression von HMOX1, einem ROS-regulierendes Protein, über eine Bindung an den Transkriptionsfaktor SP1, reguliert. Dieses transkriptionelle Regulationsnetzwerk scheint bei Prostatakrebs gestört zu sein, was möglicherweise eine zentrale Rolle beim malignen Prozess der Tumorentstehung spielt. Zusätzlich zu seiner Funktion bei der transkriptionellen Regulation gibt es bereits einige Hinweise, die eine Rolle von BRD4 bei dem zellulären Spleißprozess wahrscheinlich machen. Im Rahmen meiner Arbeit konnte ich zeigen, dass BRD4 eine wichtige Rolle bei dem Spleißvorgang unter Hitzestress spielt. So fördert es das, unter Hitzestress beeinträchtigte, Herausschneiden von Introns. Weitere molekularbiologische Analysen zeigten, dass unter diesen Stressbedingungen BRD4 in sub-nukleären Strukturen, den sogenannten „nuclear stress bodies“, rekrutiert wird. Dort aktiviert BRD4, zusammen mit dem Hitzeschock Faktor HSF1, die Transkription von nicht-kodierenden Sat III RNAs. Diese werden als wichtige Modulatoren der Stressinduzierten Spleißreaktion diskutiert. Zusammenfassend konnte ich zeigen, dass BRD4 sowohl in die Transkiption, als auch in den Spleissprozess unter zellulärem Stress involviert ist. Dies stellt eine weitere Grundlage dar, Pathomechanismen der Tumorentstehung besser zu verstehen, aber auch, um neue Therapieansätze zu entwickeln.The cellular stress response describes the adaptation of an organism to environmental stressors by a variety of molecular changes. Deregulation of this response is an indicator and possible promoter of many diseases, in particular cancers, and therefore offers an interesting target for tumor therapies. A for the tumor therapy very promising target is the bromodomains containing protein 4 (BRD4). BRD4 plays a significant role in many cellular processes: It is an epigenetic reader and transcriptional regulator and therefore links the transcription process to epigenetic patterns. The aim of this study was to further understand the role of BRD4 in the epigenetic and transcriptional regulation of cellular stress responses. Through genome-wide gene expression profiling in BRD4-deficient cells, and chromatin-protein interaction studies, I was able to identify 52 BRD4-regulated target genes, mainly encoded for proteins of the oxidative stress - and heat stress response. Further analyses highlighted BRD4 as regulator of the oxidative stress-induced KEAP1/NRF2 signalling pathway. By regulating the transcription of KEAP1, BRD4 modulates the activity of the transcription factor NRF2 and, in turn, the expression of cyto-protective genes under stress. An inhibition of BRD4 resulted in decreased reactive oxygen species (ROS) production and protected cells from oxidative stress mediated cell death. In addition, BRD4 also interacts with the transcription factor SP1 and directly regulates the expression of HMOX1, a ROS reducing protein. Remarkably, this regulatory network is disrupted in prostate cancer and thus might play a central role in tumorigenesis. Furthermore, using RNA-sequencing analyses of BRD4-deficient and heat treated cells I showed that a reduction of BRD4 expression increased the heat shock-mediated splicing inhibition, in particular intron retentions. Subsequent experiments revealed that under heat stress BRD4 binds to the heat shock factor 1 (HSF1), which leads to the recruitment of BRD4 to sub-nuclear structures, the socalled "nuclear stress bodies". The translocation of BRD4 is associated with the transcriptional activation of non-coding Sat III RNA expression. Sat III RNAs, in turn, are discussed as important modulators of the stress-induced splicing process. Taken together, my results link BRD4 not only to the transcription machinery, but also to the splicing process under oxidative or heat stress, respectively. This gives additional insights into the mode of action of BRD4 inhibitors and could lay the foundation for the development of new therapeutic strategies

Evaluation of a Prognostic Epigenetic Classification System in Chronic Lymphocytic Leukemia Patients

BACKGROUND: Methylation at 5 CpG sites was previously shown to classify chronic lymphocytic leukemia (CLL) into 3 prognostic subgroups. Here, we aimed to validate the marker set in an additional cohort and to evaluate its clinical utility for CLL patient stratification. METHODS: We evaluated this epigenetic marker set in 79 German patients using bisulfite treatment followed by pyrosequencing and classification using a support vector machine-learning tool. RESULTS: The n-CLL, i-CLL, and m-CLL classification was detected in 28 (35%). 10 (13%), and 41 (51%) patients, respectively. Epigenetic grouping was associated with IGHV mutational status (P=2 x 10(-12)), isolated del13q (P=9x 10(-6)), del17p (P= .015), complex karyotype (P= .005), VH-usage, and clinical outcome as time to first treatment (P= 1.4 x 10(-12)) and overall survival (P= .003). Multivariate Cox regression analysis identified n-CLL as a factor for earlier treatment hazard ratio (HR), 6.3 (95% confidence interval [CI] 2.4-16.4; P= .0002) compared to IGHV mutational status (HR 4.6, 95% CI 1.9-11.3, P= .0008). In addition, when comparing the prognostic value of the epigenetic classification system with the IGHV classification, epigenetic grouping performed better compared to IGHV mutational status using Kaplan-Meier estimation and allowed the identification of a third, intermediate (i-CLL) group. Thus, our study confirmed the prognostic value of the epigenetic marker set for patient stratification in routine clinical diagnostics

Designer Receptors for Nucleotide-Resolution Analysis of Genomic 5-Methylcytosine by Cellular Imaging

We report programmable receptors for the imaging-based analysis of 5-methylcytosine (5mC) in user-defined DNA sequences of single cells. Using fluorescent transcription-activator-like effectors (TALEs) that can recognize sequences of canonical and epigenetic nucleobases through selective repeats, we imaged cellular SATIII DNA, the origin of nuclear stress bodies (nSB). We achieve high nucleobase selectivity of natural repeats in imaging and demonstrate universal nucleobase binding by an engineered repeat. We use TALE pairs differing in only one such repeat in co-stains to detect 5mC in SATIII sequences with nucleotide resolution independently of differences in target accessibility. Further, we directly correlate the presence of heat shock factor 1 with 5mC at its recognition sequence, revealing a potential function of 5mC in its recruitment as initial step of nSB formation. This opens a new avenue for studying 5mC functions in chromatin regulation in situ with nucleotide, locus, and cell resolution

The bromodomain protein BRD4 regulates splicing during heat shock

The cellular response to heat stress is an ancient and evolutionarily highly conserved defence mechanism characterised by the transcriptional up-regulation of cyto-protective genes and a partial inhibition of splicing. These features closely resemble the proteotoxic stress response during tumor development. The bromodomain protein BRD4 has been identified as an integral member of the oxidative stress as well as of the inflammatory response, mainly due to its role in the transcriptional regulation process. In addition, there are also several lines of evidence implicating BRD4 in the splicing process. Using RNAsequencing we found a significant increase in splicing inhibition, in particular intron retentions (IR), following heat treatment in BRD4-depleted cells. This leads to a decrease of mRNA abundancy of the affected transcripts, most likely due to premature termination codons. Subsequent experiments revealed that BRD4 interacts with the heat shock factor 1 ( HSF1) such that under heat stress BRD4 is recruited to nuclear stress bodies and non-coding SatIII RNA transcripts are up-regulated. These findings implicate BRD4 as an important regulator of splicing during heat stress. Our data which links BRD4 to the stress induced splicing process may provide novel mechanisms of BRD4 inhibitors in regard to anticancer therapies

Loss of Msh2 and a single-radiation hit induce common, genome-wide, and persistent epigenetic changes in the intestine

BackgroundMismatch repair (MMR)-deficiency increases the risk of colorectal tumorigenesis. To determine whether the tumors develop on a normal or disturbed epigenetic background and how radiation affects this, we quantified genome-wide histone H3 methylation profiles in macroscopic normal intestinal tissue of young radiated and untreated MMR-deficient VCMsh2(LoxP/LoxP) (Msh2(-/-)) mice months before tumor onset.ResultsHistone H3 methylation increases in Msh2(-/-) compared to control Msh2(+/+) mice. Activating H3K4me3 and H3K36me3 histone marks frequently accumulate at genes that are H3K27me3 or H3K4me3 modified in Msh2(+/+) mice, respectively. The genes recruiting H3K36me3 enrich in gene sets associated with DNA repair, RNA processing, and ribosome biogenesis that become transcriptionally upregulated in the developing tumors. A similar epigenetic effect is present in Msh2(+/+) mice 4weeks after a single-radiation hit, whereas radiation of Msh2(-/-) mice left their histone methylation profiles almost unchanged.ConclusionsMMR deficiency results in genome-wide changes in histone H3 methylation profiles preceding tumor development. Similar changes constitute a persistent epigenetic signature of radiation-induced DNA damage

Co-inhibition of BET proteins and PI3K alpha triggers mitochondrial apoptosis in rhabdomyosarcoma cells

Remodeling transcription by targeting bromodomain and extraterminal (BET) proteins has emerged as promising anticancer strategy. Here, we identify a novel synergistic interaction of the BET inhibitor JQ1 with the PI3K alpha-specific inhibitor BYL719 to trigger mitochondrial apoptosis and to suppress tumor growth in models of rhabdomyosarcoma (RMS). RNA-Seq revealed that JQ1/BYL719 co-treatment shifts the overall balance of BCL-2 family gene expression towards apoptosis and upregulates expression of BMF, BCL2L11 (BIM), and PMAIP1 (NOXA) while downregulating BCL2L1 (BCL-x(L)). These changes were confirmed by qRT-PCR and western blot analysis. Ingenuity pathway analysis (IPA) of RNA-Seq data followed by validation qRT-PCR and western blot identified MYC and FOXO3a as potential transcription factors (TFs) upstream of the observed gene expression pattern. Immunoprecipitation (IP) studies showed that JQ1/BYL719-stimulated increase in BIM expression enhances the neutralization of antiapoptotic BCL-2, BCL-x(L), and MCL-1. This promotes the activation of BAK and BAX and caspase-dependent apoptosis, as (1) individual silencing of BMF, BIM, NOXA, BAK, or BAX, (2) overexpression of BCL-2 or MCL-1 or (3) the caspase inhibitor N-Benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethylketone (zVAD.fmk) all rescue JQ1/BYL719-induced cell death. In conclusion, co-inhibition of BET proteins and PI3K alpha cooperatively induces mitochondrial apoptosis by proapoptotic re-balancing of BCL-2 family proteins. This discovery opens exciting perspectives for therapeutic exploitation of BET inhibitors in RMS