Dynamic chromatin: concerted nucleosome remodelling and acetylation

The flexibility of chromatin that enables translation of environmental cues into changes in genome utilisation, relies on a battery of enzymes able to modulate chromatin structure in a highly targeted and regulated manner. The most dynamic structural changes are brought about by two kinds of enzymes with different functional principles. Changes in the acetylation status of histones modulate the folding of the nucleosomal fibre. The histone-DNA interactions that define the nucleosome itself can be disrupted by ATP-dependent remodelling factors. This review focuses on recent developments that illustrate various strategies for integrating these disparate activities into complex regulatory schemes. Synergies may be brought about by consecutive or parallel action during the stepwise process of chromatin opening or closing. Tight co-ordination may be achieved by direct interaction of (de-)acetylation enzymes and remodelling ATPases or even permanent residence within the same multi-enzyme complex. The fact that remodelling ATPases can be acetylated by histone acetyltransferases themselves suggests exciting possibilities for the coordinate modulation of chromatin structure and remodelling enzymes

Regulation of β-Casein Gene Expression by Octamer Transcription Factors and Utilization of β-Casein Gene Promoter to Produce Recombinant Human Proinsulin in the Transgenic Milk

β-Casein is a major milk protein, which is synthesized in mammary alveolar secretory epithelial cells (MECs) upon the stimulation of lactogenic hormones, mainly prolactin and glucocorticoids (HP). Previous studies revealed that the proximal promoter (-258 bp to +7 bp) of the β-casein gene is sufficient for induction of the promoter activity by HP. This proximal region contains the binding sites for the signal transducer and activator of transcription 5 (STAT5), glucocorticoid receptor (GR), and octamer transcription factors (Oct). STAT5 and GR are essential downstream mediators of prolactin and glucocorticoid signaling, respectively. This study investigated the functions of Oct-1 and Oct-2 in HP induction of β-casein gene expression. By transiently transfection experiment, we showed that individual overexpression of Oct-1 and Oct-2 further enhanced HP-induced β-casein promoter activity, respectively, while Oct-1 and Oct-2 knockdown significantly inhibited the HP-induced β-casein promoter activity, respectively. HP rapidly induced the binding of both Oct-1 and Oct-2 to the β-casein promoter, and this induction was not mediated by either increasing their expression or inducing their translocation to the nucleus. In MECs, Oct-2 was found to physically interact with Oct-1 regardless of HP treatment. However, HP induced physical interactions of Oct-1 or Oct-2 with both STAT5 and GR. Although the interaction between Oct-1 and Oct-2 did not synergistically stimulate HP-induced β-casein gene promoter activity, the synergistic effect was observed for the interactions of Oct-1 or Oct-2 with STAT5 and GR. The interactions of Oct-1 with STAT5 and GR enhanced or stabilized the binding of STAT5 and GR to the promoter. Abolishing the interaction between Oct-1 and STAT5 significantly reduced the hormonal induction of β-casein gene transcription. Thus, our study indicates that HP activate β-casein gene expression by inducing the physical interactions of Oct-1 and Oct-2 with STAT5 and GR in mouse MECs. There is a high and increasing demand for insulin because of the rapid increase in diabetes incidence worldwide. However, the current manufacturing capacities can barely meet the increasing global demand for insulin, and the cost of insulin production keeps rising. The mammary glands of dairy animals have been regarded as ideal bioreactors for mass production of therapeutically important human proteins. We tested the feasibility of producing human proinsulin in the milk of transgenic mice. In this study, four lines of transgenic mice were generated to harbor the human insulin gene driven by the goat β-casein gene promoter. The recombinant human proinsulin was detected in the milk by Western blotting and enzyme-linked immunosorbent assay. The highest expression level of human proinsulin was as high as 8.1 μg/µl in milk of transgenic mice at mid-lactation. The expression of the transgene was only detected in the mammary gland during lactation. The transgene expression profile throughout lactation resembled the milk yield curve, with higher expression level at middle lactation and lower expression level at early and late lactation. The blood glucose and insulin levels and major milk compositions of transgenic mice were not changed. The mature insulin derived from the milk proinsulin retained biological activity. Thus, our study indicates that it is practical to produce high levels of human proinsulin in the milk of dairy animals, such as dairy cattle and goat

Epigenetics, Behaviour, and Health

<p/> <p>The long-term effects of behaviour and environmental exposures, particularly during childhood, on health outcomes are well documented. Particularly thought provoking is the notion that exposures to different social environments have a long-lasting impact on human physical health. However, the mechanisms mediating the effects of the environment are still unclear. In the last decade, the main focus of attention was the genome, and interindividual genetic polymorphisms were sought after as the principal basis for susceptibility to disease. However, it is becoming clear that recent dramatic increases in the incidence of certain human pathologies, such as asthma and type 2 diabetes, cannot be explained just on the basis of a genetic drift. It is therefore extremely important to unravel the molecular links between the "environmental" exposure, which is believed to be behind this emerging incidence in certain human pathologies, and the disease's molecular mechanisms. Although it is clear that most human pathologies involve long-term changes in gene function, these might be caused by mechanisms other than changes in the deoxyribonucleic acid (DNA) sequence. The genome is programmed by the epigenome, which is composed of chromatin and a covalent modification of DNA by methylation. It is postulated here that "epigenetic" mechanisms mediate the effects of behavioural and environmental exposures early in life, as well as lifelong environmental exposures and the susceptibility to disease later in life. In contrast to genetic sequence differences, epigenetic aberrations are potentially reversible, raising the hope for interventions that will be able to reverse deleterious epigenetic programming.</p

Linker Histone H1 and Androgen Receptor: Two Different Players in the Chromatin Orchestra

The linker histone H1 and the androgen receptor are two different players in the chromatin orchestra. The linker histone H1, one of the most abundant proteins in the nucleus, is located at the surface of the nucleosome but despite many important functions reported for this protein it is not as well studied as the core histones. The androgen receptor, AR, is a member of the nuclear receptor family, a conserved family of transcription factors. AR is of uttermost importance for many functions in the human body as well as a driving force behind the most common cancer form in Sweden: prostate cancer. Paper I: Here we focus on the linker histone and the question of whether the heterogeneity of the linker histone family has a functional significance. By reconstituting individual H1 subtypes in Xenopus oocytes, a model system that lacks somatic linker histone, we have systematically studied their specific binding to chromatin and their effect on the chromatin structure as seen by increase in nucleosomal repeat length, NRL. We have compared linker histones that differ both in terms of origin and expression pattern as well as the ubiquitously expressed human somatic subtypes. We show that the biggest differences in terms of effect on chromatin structure are found between the coexisting human subtypes thus suggesting that H1 subtypes have different roles in the organization and function of the chromatin fiber. Paper II: Previous studies have shown that the binding abilities of H1 are at large determined by the properties of its C-terminal domain while much less attention has been paid to the role of the N-terminal domain. Using the same assay as in Paper I we compared the binding properties of wild type H1.4 and hH1.4 devoid of its N-terminal domain (ΔN-hH1.4). We showed that the lack of N-terminal domain does not have any effect on the hH1.4 induced increase in the NRL; however, the ΔN-hH1.4 displays a drastically lower affinity for chromatin binding as compared to the wt hH1.4 and is more prone to unspecific chromatin binding. We conclude that the Nterminal domain of H1 is an important determinant of affinity and specificity of H1-chromatin interactions. Paper III: Prostate cancer growth is regulated by AR. Antiandrogens (AR antagonists) compete with androgens for binding to AR and are thus used to stall cancer cells. However, invariably patients develop resistance to such therapy and relapse with castration-resistant prostate cancer. This motivates the creation of a second generation of AR antagonists with a more clear-cut anti AR activity. By reconstitution of the hormone regulated mouse mammary tumor virus promoter, MMTV, in Xenopus oocytes we previously revealed that the transcription factor FoxA1 is able to convert the glucocorticoid antagonist RU-486 to a partial agonist by presetting of the chromatin structure at the hormone-responsive enhancer. High level of FoxA1 is a negative prognostic factor in prostate cancer and we decided to evaluate the effect of the AR antagonists bicalutamide (BIC) and MDV3100 (MDV) on transcriptional outcome of AR-dependent MMTV promoter in the context of FoxA1. Here we show that both antagonists, upon binding to AR, can translocate the AR-ligand complex to the nucleus, albeit with reduced efficiency for MDV. While in the nucleus both AR-antagonist complexes have the potential to bind sequence specifically to the hormone response elements, HREs, in vivo. The DNA binding is strongly enhanced by co-expression of FoxA1 that makes the HREs more accessible for AR binding. In this context BIC antiandrogenic ability is seriously compromised whereas MDV shows a more persistent antagonistic activity. We believe that these findings may be of clinical relevance

STAT5-Interacting proteins: A synopsis of proteins that regulate STAT5 activity

© 2017 by the authors. Licensee MDPI, Basel, Switzerland. Signal Transducers and Activators of Transcription (STATs) are key components of the JAK/STAT pathway. Of the seven STATs, STAT5A and STAT5B are of particular interest for their critical roles in cellular differentiation, adipogenesis, oncogenesis, and immune function. The interactions of STAT5A and STAT5B with cytokine/hormone receptors, nuclear receptors, transcriptional regulators, proto-oncogenes, kinases, and phosphatases all contribute to modulating STAT5 activity. Among these STAT5 interacting proteins, some serve as coactivators or corepressors to regulate STAT5 transcriptional activity and some proteins can interact with STAT5 to enhance or repress STAT5 signaling. In addition, a few STAT5 interacting proteins have been identified as positive regulators of STAT5 that alter serine and tyrosine phosphorylation of STAT5 while other proteins have been identified as negative regulators of STAT5 via dephosphorylation. This review article will discuss how STAT5 activity is modulated by proteins that physically interact with STAT5

Funktionelle Charakterisierung der C-terminalen-Domänen des Korepressors N-CoR

Although in general cells are genetically identical in multicellular organisms, the differential expression of genomic information enables cell type definition and specific organ function. In eukaryotic cells, the DNA is associated with histone and non-histones proteins into a restrictive structure called chromatin. Assembly into chromatin does not only protect and package the linear double stranded DNA into the nucleus but is fundamental for the execution of diverse genetic programs. Posttranslational modifications of histones regulate the accessibility of the DNA to transcription factors and serve as scaffold for binding of regulatory proteins. Nuclear receptors are transcription factors that bind specific target sequences on the DNA and recruit transcriptional coregulators at the promoter. These are able to modify the chromatin structure in an activating or repressing manner. The contribution of corepressors to the biological actions of nuclear receptors has turned out to be essential. Impaired corepressor function can be the cause of endocrine malfunctions, neoplastic diseases or severe developmental abnormalities. To better understand the role of the nuclear receptor corepressor N-CoR the unknown function of the extreme C-terminus was investigated. In this thesis the interaction of N-CoR with the non-POU-domain containing octamer-binding protein Non0/p54nrb, that was found tobe a potential interaction partner in a yeast-two-hybrid screen, was confirmed. This protein contains two RNA recognition motifs (RRM) and is described as a multifunctional protein since it is involved in transcription Initiation as well as in pre-mRNA processing. The RRM1 motif was determined to be essential and sufficient for the interaction with N-CoR. Obtaining dominant negative effect with the Non0/p54nrb RRM1 deletion mutant in functional reporter assays, data support that NonO modulates the capacity of N-CoR to repress and alters the recruitment of N-CoR by nuclear receptors to targeted Promoters. Additional analyses suggest that the N- and C- terminus of N-CoR are involved in intramolecular interactions and that they regulate each other. Taken results together a functional model is proposed that supports the biological relevance of the interaction of N-CoR with NonO and the function of N-CoR C-terminus acting as asensor that evaluates the ratio of corepressors and coactivators in the nuclear receptor environment. N-CoR repressive capacity would be altered by modulating factors like NonO that interacts with N-CoR C-terminus. The mechanism support that splicing and transcription regulation are physically and functionallylinked to ensure the appropriate amount of messager RNA to be transcript and process in response to stimulation intensity and cell context.Die fehlerhafte Rekrutierung von Korepressoren wie dem Protein N-CoR (nuclear receptor co-repressor) ist die Ursache von genetischer Krankheiten und verschiedener Leukämien. Darüber hinaus ist N-CoR vermittelte Repression für die Entwicklung von Säugern entscheidend. N-CoR Knockoutmäuse sterben in der mittleren Phase der Embryonalentwicklung und zeigen Defekte in der Reifung von Erythrozyten, Thymozyten und in der Entwicklung verschiedener neuronaler Strukturen. Dies ist zum Teil auf die Störung der Repression zurückzuführen, die von nuklearen Hormonrezeptoren wie Retinsäure- und Thyroidhormonrezeptoren vermitteltet wird. N-CoR ist in der Zelle mit Histondeacetylasen (HDACs) komplexiert. Diese Enzyme bewirken im Zusammenspiel mit den Histonacetyltransferasen durch Deacetylierung beziehungsweise Acetylierung von Histonen eine dynamische Modifikation des Chromatins und beeinflussen so die Transkription von Genen, Obwohl N-CoR ein seit längerer Zeit bekanntes Protein ist, enthält es bislang uncharakterisierte Domänen wie die äußerste carboxyterminale Region. Diese Domäne wurde als Köder in einem Hefe-Zwei-Hybrid Screen zur Identifizierung von Interaktionspartnern des N-CoR C-Terminus eingesetzt. In der vorliegenden Arbeit wurde die Interaktion von N-CoR mit dem multifunktionellen Protein Non0/p54nrb (non POU domain containing octamer binding protein) bestätigt und die biologische Bedeutung dieser Interaktion wurde untersucht. Non0/p54nrb ist an verschiedenen Prozessen wie der RNA Polymerase II Komplexbildung und -aktivierung, Splicesosom Komplexbildung und der Bildung des RNA Polyadenylierungskomplexes beteiligt. Dieses RNA-Erkennungsmotive (RRM) enthaltende Protein spielt eine regulatorische Rolle in der Hormonrezeptor- abhängigen Transkriptionsregulation. Es wurde festgestellt, dass das RRM1 Motiv des Proteins Non0/p54nr für die Interaktion mit N-CoR entscheidend ist und, dass NonO sowohl die Interaktion zwischen nuklearen Rezeptoren und N-CoR als auch die N-CoR Repressionsaktivität beeinflussen kann. Ferner wurde gezeigt, dass der extreme N-CoR C- Terminus möglicherweise eine regulierende Domäne ist, welche die N-CoR Repressionsaktivität des Amino-Terminus modulieren kann. Das vorgeschlagene funktionelle Modell stellt ein bislang nicht bekanntes Regulationselement in der Kontrolle der Genexpression dar. N-CoR repressive Kapazität und die Rekrutierung des Korepressors zu Promotorenregionen wäre nicht nur von dem Aktivierungsstandder Nuklearen Rezeptoren abhängig aber auch von dem Verhältnis von Koaktivator und Korepressor in der Umgebung der Nuklearen Rezeptoren und von den Interaktionen mit modulierende Protein wie Non0/p54nrb welche die physische Verbindung zwischen transkriptionsakivierenden und -reprimierenden Prozessen sein könnte

Dynamic regulation of glucocorticoid signalling in health and disease

Activation of the glucocorticoid receptor (GR) by endogenous and synthetic glucocorticoids regulates hundreds of genes to control regulatory networks in development, metabolism, cognition and inflammation. Elucidation of the mechanisms that regulate glucocorticoid action has highlighted the dynamic nature of hormone signalling and provides novel insights into genomic glucocorticoid actions. The major factors that regulate GR function include chromatin structure, epigenetics, genetic variation and the pattern of glucocorticoid hormone secretion. We review our current understanding of the mechanisms that contribute to GR signalling and how these contribute to glucocorticoid sensitivity, resistance and side effects

Remodelatge de la cromatina durant la inducció per progesterona del promotor de l'MMTV

Per a comprendre els mecanismes que governen l'expressió dels gens inclosos en els genomes eucariòtics és necessari prendre en consideració la manera en què les regions reguladores d'aquests gens estan organitzades en la cromatina. Les diferències en l'organització de la cromatina i en les modificacions químiques dels components de la cromatina expliquen els diferents patrons de l'expressió gènica en diversos tipus de cèll. ules i la seva resposta específica a senyals externs. Basant-nos en els nostres estudis sobre la inducció hormonal del promotor del mouse mammary tumour virus (MMTV), podem concloure que la seqüència nucleotídica primària determina no solament la manera en què la doble hèlix de DNA envolta l'octàmer d'histona, i així l'accessibilitat de punts d'unió per als factors de transcripció, sinó també la manera en què aquests factors estableixen sinergismes i la naturalesa del remodelatge de la cromatina dependent d'ATP. A més, la senyalització via crosstalk amb cascades de cinases citoplasmàtiques canvia l'estructura de la cromatina en els gens diana i és fonamental per a la correcta regulació a través de receptors d'hormones esteroidees.Understanding the mechanisms governing the expression of the genes encompassed in the eukaryotic genomes requires a careful consideration of the way regulatory regions of these genes are packaged in chromatin. Differences in the chromatin organization and in the chemical modifications of chromatin components account for the different patterns of gene expression in various cell types and for their specific response to external signals. Based on our studies on the hormonal induction of mouse mammary tumour virus (MMTV) promoter we conclude that the primary nucleotide sequence determines not only the way theDNAdouble helix wraps around the histone octamer, and so the accessibility of binding sites for transcription factors, but also the way these factors synergize and the nature of the ATP-dependent chromatin remodelling. Moreover, signalling via crosstalk with cytoplasmic kinase cascades changes the chromatin structure of target genes and is essential for proper regulation by steroid hormone receptors