A fim de contornar a problemática imposta pela recalcitrância da parede celular à hidrólise, o processo de produção de etanol a partir de biomassa vegetal requer um passo denominado de pré-tratamento, que consiste em tornar a biomassa mais acessível à ação de glicosil hidrolases que atacam a parede celular. O melhor conhecimento de processos de degradação de parede operados pela própria planta tem o potencial de direcionar as pesquisas em bioernegia, indicando quais os mecanismos mais eficientes para desmontar o complexo de polímeros da parede celular. Cunhado pré-tratamento biológico, este consiste na hidrólise de polissacarídeos estruturais empregando mecanismos e elementos chave de processos de degradação de parede celular que ocorrem na própria planta. Um exemplo de evento com esta característica é a formação de aerênquima lisígeno, que consiste na abertura de espaços de gás em tecidos parenquimáticos. A formação do aerênquima em cana-de-açúcar se dá de forma modular, sendo o processo constituído por seis etapas: 1) percepção do sinal inicial; 2) separação celular: 3) expansão celular; 4) morte celular programada; 5) hidrólise de hemiceluloses e 6) hidrólise de celulose. O presente trabalho teve como principal foco estudar a regulação das duas etapas iniciais, visando obter conhecimentos que possibilitem para tornar a biomassa de cana de açúcar menos resistente à penetração de enzimas. O aerênquima ocorre na raiz de cana-de-açúcar por um processo constitutivo. Sua independência de um indutor externo foi corroborada mediante tratamento com nutrientes e inibidor da percepção por etileno (1-MCP) pela análise dos cinco centímetros mais apicais da raiz. O atraso na formação do aerênquima após tratamento com nutrientes levou à expressão diferencial de genes relacionados à degradação de parede celular, morte celular programada e sinalização por etileno. Por outro lado, o 1-MCP não afetou visivelmente a formação do aerênquima, porém alterou o balanço hormonal na raiz. O padrão transcricional das raízes tratadas com 1-MCP revelou aumento da expressão de genes relacionados à expansão celular e estresse oxidativo. Tais padrões são discutidos à luz da regulação hormonal da formação do aerênquima através do estabelecimento de um balanço hormonal definido entre auxina e etileno. Ambos os experimentos levaram à seleção de quatro genes candidatos: dois fatores de transcrição (ScRAV1 e ScERF1) e duas glicosil hidrolases (ScEPG1 e ScARA1), sequenciados e analisados quanto à diversidade hom(e)óloga, sequências promotoras e estrutura gênica em comparação a S. bicolor. A topologia das reconstruções filogenéticas e a distribuição diferencial de sítios para RAV e ERF nos promotores de ScEPG1 e ScARA1 não parece refletir padrões de expressão distintos, visto que os diferentes hom(e)ólogos demonstraram ser igualmente expressos. Em sistema heterólogo, o fator de transcrição RAV apresentou atividade repressora sobre o promotor de ScEPG1. Tal papel de RAV sugere regulação negativa sobre a degradação da lamela média e sobre o processo de perda de adesão e separação celular. A identificação de reguladores-chave da formação do aerênquima consiste em importante elo entre a sinalização hormonal e a degradação de parede celular, possibilitando a melhor compreensão dos mecanismos de controle da degradação endógena de parede celular. Os dados produzidos possibilitam a aplicação biotecnológica dos genes sequenciados, além de consistirem em significativo avanço no conhecimento sobre a fisiologia do processo estudado e na dinâmica da regulação da expressão gênica acoplada a aspectos filogenéticos das sequências alvo. / In order to circumvent the problems imposed by the cell wall recalcitrance to hydrolysis, the process underlying biofuel production requires a step called pretreatment, aimed at making biomass more accessible to the action of glycosil hydrolases that attack the cell wall. The increased knowledge of wall degradation processes operated by the plant itself has the potential to influence research in the bioernergy field, pointing out the most efficient mechanisms to disassemble the cell wall complex polymeric structure. The term coined biological pretreatment means taking advantage of key elements and mechanisms of cell wall degradation processes occurring in the plant itself in order to disassemble the entangled polysaccharide structure. One example of endogenous cell wall degradation process is the lysigenous aerenchyma formation, which consists in the opening of gas spaces in parenchyma tissue. The aerenchyma formation in sugarcane is thought to be a modular process that occurs in six steps: 1) target cells perception; 2) cell separation; 3) cell expansion; 4) programmed cell death; 5) hemicellulose hydrolysis and 6) cellulose hydrolysis. This work has as the main objective to study the regulation of the two initial steps, aiming at acquiring knowledge that would afford to turn biomass less resistant to enzyme penetration. The aerenchyma develops within the roots of sugarcane as a constitutive process. Its independence from an external inducer was corroborated in this work by subjecting sugarcane plants to treatment with nutrients and one inhibitor of ethylene perception (1-MCP) when the five more apical centimeters of the root were analyzed. After treatment with nutrients, the delayed aerenchyma formation led to differential expression of cell wall degradation-, programmed cell death- and ethylene signalling-related genes. Under visual inspection, 1-MCP did not show effect on aerenchyma development. Instead, it changed the hormone balance mainly in the two most apical root segments. The transcriptional pattern of 1-MCP treated roots revealed increased expression of cell expansion- and oxidative stress-related genes. Such patterns are discussed in the light of the hormone regulation of the aerenchyma development through the establishment of a balance between auxin and ethylene within root segments. Both experiments led to the selection of four two candidate genes, two transcription factors (ScRAV1 and ScERF1) and two glycosil hydrolases (ScEPG1 and ScARA1), that were sequenced and analyzed regarding homEURologous diversity, promoter sequences and gene structure compared to S. bicolor. The topology of the phylogenetic reconstructions and the differential distribution of sites for RAV and ERF within ScEPG1 and ScARA1 promoters did not reflect distinct expression patterns. Different hom(e)ologous of each target gene were equally expressed. In an heterologous system, RAV transcription factor interacted with ScEPG1 promoter, reducing the activity encoded by the reporter gene. This suggests the repressive role of RAV on pectin degradation within the middle lamella, leading to reduced cell adhesion and cell separation, possibly regulated by this glycosyl hydrolase. The identification of key regulators of the aerenchyma formation is an important link between hormone signaling and the wall degradation within the sugarcane roots. It enables a better understanding of control mechanisms underlying wall modifications when performed by the plant itself. Altogether, the data produced in this work allows biotechnological application of sequenced genes. Moreover, the produced data unveil key aspects regarding physiologycal aspects of aerenchyma development and highlights features related to the dynamics of gene expression in sugarcane coupled to the phylogenetic aspects of the four target sequences.

Nesta tese propomos um modelo estocástico multimodal para regulação da expressão gênica em nível de transcrição. A definição de um espaço de parâmetros que contém o conteúdo biológico do sistema aliada à escolha apropriada de uma base para construir a matriz de acoplamento entre os estados do sistema levaram à obtenção de soluções exatas do modelo. Tais soluções são obtidas transformando as equações mestras em equações diferenciais parciais usando a técnica das funções geradoras e escrevendo os coeficientes das equações parciais em termos dos parâmetros biológicos do modelo. No regime estacionário obtivemos uma relação de recorrência para os coeficientes das séries de potências que definem as funções geradoras e a especificação das configurações de equilíbrio do sistema permite que estas séries sejam calculadas exatamente. Com as soluções exatas calculadas não só as distribuições de probabilidade foram obtidas como os momentos das distribuições. As distribuições de probabilidade de equilíbrio apresentam estruturas multimodais com vários picos e a análise do ruído (flutuação) mostra que a existência de um estado intermediário de eficiência transcricional leva a redução do ruído global do sistema. A inspeção dos autovalores da matriz de acoplamento mostrou que existem regiões onde a dinâmica dos momentos é de caráter oscilatória com amortecimento. Diferentes esquemas de acoplamento levam à diferentes regimes transientes, tal característica revela que o sistema multimodal apresentam maior flexibilidade adaptativa quando comparado com sistemas de um ou dois estados. / In this thesis we propose a stochastic model for multimodal regulation of gene expression at the transcriptional level. The definition of a parameter space that contains the contents of the biological system coupled with the appropriate choice of a base to build the coupling matrix between the states of the system led to the exact solutions of the model. Such solutions are obtained by transforming master equations in partial differential equations using the technique of generating functions and writing the coefficients of partial equations in terms of biological parameters of the model. In the steady a recurrence relation for the coefficients of power series defining the generating functions was obtained and specification of the equilibrium configurations of the system allows the exact calculation of these series. With the exact solutions calculated not only the probability distributions were obtained but also the moments of the distributions. The equilibrium distributions probability is multimodal and presents several peaks. Analysis of the noise (fluctuation) shows that the existence of an state with intermediate transcriptional efficiency leads to a reduction of the overall system noise. Inspection of the eigenvalues of the coupling matrix showed that there are regions where the dynamics of the moments is damped oscillating. Different coupling schemes lead to different transient regimes, this feature reveals that the multimodal system have greater adaptive flexibility when compared to systems of one or two states.

Le développement embryonnaire précoce des mammifères femelles s’accompagne de l’inactivation transcriptionnelle d’un de leurs deux chromosomes X. Cet évènement est initié suite à l’expression mono-allélique de l’ARN non codant Xist, qui est contrôlée par de nombreux éléments cis-régulateurs présents dans le centre d’inactivation du chromosome X (Xic) – tel son anti-sens répresseur Tsix. Mon travail de thèse a consisté à développer des approches permettant d’appréhender le paysage structural dans lequel s’exerce cette régulation. La caractérisation de l’architecture tridimensionnelle du Xic, par des techniques basées sur la capture de conformation chromosomique (3C) et l’hybridation in situ en fluorescence (FISH), m’a permis de mettre en évidence que les promoteurs respectifs de Xist et Tsix sont engagés dans des interactions physiques intimes avec des loci distaux, localisés au sein du Xic, et de montrer qu’au moins certaines de ces régions exercent un effets régulateurs à longue-distance. Les éléments du Xic contactés par les régions promotrices de Xist et de Tsix sont en outre fondamentalement différents, chacune engageant des associations chromosomiques sur plusieurs centaines de kilobases dans leur direction 5’ respective.Ce travail a également permis de révéler des propriétés insoupçonnées de l’architecture chromosomiques. En effet, le Xic apparaît scindé en plusieurs sous-régions, couvrant chacune entre 200kb et 1Mb, à l’intérieur desquelles les interactions chromosomiques sont préférentiellement établies. L’existence de ces domaines d’interaction s’intègre avec d’autres propriétés structurales du génome, tels la composition de la chromatine sous-jacente et l’association à la lamine nucléaire, mais n’apparaît pas en dépendre directement. En étudiant la dynamique de la conformation chromosomique du Xic au cours de la différenciation cellulaire, j’ai pu constater la robustesse de cette organisation, sauf sur le chromosome X inactif, qui se distingue par la perte des contacts chromosomiques préférentiels détectables sur son homologue actif.Enfin, j’ai pu mettre en évidence que la variabilité du repliement général du chromosome X amène à un instant donné chaque allèle de Tsix à contacter physiquement des jeux de séquences distales différents, suggérant que l’environnement structural instantané de chacun de ces allèles à l’orée de l’activation mono-allélique de Xist est différent. Ce travail, combinant des approches à l’échelle de la population cellulaire d’une part et de la fibre de chromatine unique d’autre part, apporte une nouvelle vision du paysage structural et régulateur dans lequel s’inscrit le contrôle de l’activité transcriptionnelle de Xist, et fourni de nouvelles perspectives concernant les principes fondamentaux de l’organisation topologique des chromosomes chez les mammifères. / Early development of female mammals is accompanied by transcriptional inactivation of one of their two X chromosomes. This event is initiated following mono-allelic expression of the Xist non-coding RNA – what is achieved by the interplay of numerous cis-regulatory elements present within the X inactivation center (Xic), such as its repressive antisense Tsix. Our work aimed at throwing light on the structural landscape that underlies such long-range regulation. Characterization of the three-dimensional architecture of the Xic, by the means of Chromosome Conformation Capture (3C)-based techniques and in situ fluorescence hybridization (FISH), revealed that the respective promoters of Xist and Tsix contact many distal genomic elements within the Xic, and that at least one of such interacting region exerts long-range cis-transcriptional control. Noticeably, Xist and Tsix promoters associate with different sets of elements in their respective 5’ direction that are spread out over several hundreds of kilobases These experiments also revealed unforeseen properties of chromatin architecture. Indeed, the Xic appears to be partitioned in several sub-regions, each spanning between 200kb and 1Mb, inside which chromosomal interactions are preferentially established. The existence of these interaction domains integrates with other structural features of the genome, such as underlying chromatin composition and association with the nuclear lamina, but does not seem to directly depend on them. By analyzing chromosome conformation of the Xic during cell differentiation we document the robustness of this organizational principle, with the noticeable exception of the inactive X chromosome that assumes a folding pattern that is more random than its active homolog. Finally we also bring evidence that variability in the folding pattern of the two X chromosomes in the same cell brings each Tsix allele in association with different sets of chromosomal partners at a given moment, suggesting that the instantaneous structural environment of each allele at the onset of mono-allelic Xist up-regulation is different.By combining approaches at the scale of cell populations on the one hand, and at the single chromatin fiber level on the other, this study provides a first vision of the structural landscape in which Xist regulation takes place, and brings new insights concerning fundamental properties of chromosome organization in mammals.

No description available.

CD4+ and CD8+ T cells are critical components of the adaptive arm of immune responses. During viral infection, CD8+ T cells utilize their cytotoxic function to kill infected cells and clear the infection. In addition, CD4+ T cells differentiate into either T helper 1 (Th1) or T follicular helper (Tfh) cells, which provide essential help to enhance the efficacy of other response immune cells, including macrophages, CD8+ T cells, and B cells. The transcription factor, T cell factor-1 (TCF1), and its homologue, Lymphoid enhancer-binding factor-1 (LEF1), have critical roles in the development, differentiation, and persistence of both CD4+ and CD8+ T cells. However, the influence of TCF1 and LEF1 on Th1 and Tfh differentiation remains to be examined. Furthermore, due to alternative promoter usage, TCF1 and LEF1 are expressed as both long and short isoforms. The distinct roles of the long and short isoforms of TCF1 in the context of CD4+ and CD8+ T cell responses have not been defined. My studies utilized multiple novel mouse strains to examine the roles of TCF1 and LEF1 in Tfh and Th1 differentiation during viral infection, and the unique requirements of TCF1 long isoforms in CD4+ and CD8+ T cell responses. Specifically, my initial studies characterized a new TCF1 reporter construct (referred to as p45GFP reporter) and used this reporter to address the specific contributions of TCF1 long isoforms to the CD8+ T cell response. Previous studies have abrogated all TCF1 isoforms and shown that in the absence of TCF1, the memory CD8+ T cell population is dramatically impaired and exhibits defective persistence over time. Here, I showed that TCF1 short isoforms are sufficient for the generation of memory CD8+ T cells, however TCF1 long isoforms are important for the maturation of memory CD8+ T cells. Another critical component of pathogen clearance and long-term protection is a productive humoral response, which is optimized by the B cell help provided by Tfh cells. Using the p45GFP reporter, I showed that TCF1 is specifically retained in Tfh cells, but downregulated in Th1 cells. I utilized a huCd2-Cre system to conditionally delete TCF1 and LEF1 in mature T cells. In response to viral infection, TCF1 and LEF1 double-deficient mice showed normal Th1 responses, but severely defective Tfh differentiation and a concomitant impaired B cell response. I further demonstrated that TCF1 promotes Tfh differentiation by directly regulating many Tfh-associated genes. Furthermore, I used the p45GFP reporter to I identified distinct, but critical, roles for both long and short isoforms of TCF1 in driving Tfh differentiation and repressing differentiation toward Th1 or germinal center Tfh cells. Finally, while TCF1 is known to be critical in the formation of memory CD8+ T cells, its impact on memory CD4+ T cell generation has not been assessed. Once again utilizing the p45GFP reporter, my studies identified an important role for TCF1 long isoforms in the survival of both Th1 and Tfh cells through contraction. In the absence of TCF1 long isoforms, the memory CD4+ T cell population is severely reduced. Taken together, my work has demonstrated critical roles for TCF1 during both effector and memory phases of the CD4+ T cell response to viral infection. In summary, TCF1 is crucial for CD4+ T cells to effectively differentiate and provide important help to B cells during viral infection. Moreover, my studies have identified critical and unique roles for long and short isoforms of TCF1. Finally, TCF1 is necessary for optimal formation of memory CD4+ and CD8+ T cells, and thus is an essential component in achieving protective immunological memory after viral infection.

A 700bp portion of the promoter of CYP2D25, the porcine microsomal vitamin D 25-hydroxylase was isolated and sequenced. The computer analysis of the sequence revealed the existence of a putative VDRE at 220 bp upstream of the transcription start site. A CYP2D25 promoter-luciferase reporter plasmid was constructed in order to study the transcriptional regulation of the gene. Treatment with the vitamin D metabolites calcidiol and calcitriol suppressed the promoter, provided that the nuclear receptors VDR and RXR were overexpressed. Phenobarbital was also capable of suppressing the promoter if the nuclear receptors PXR or CAR were overexpressed. The 25-hydroxylases are not expressed solely in liver but in a wide array of other organs as well. It is therefore possible at least in theory to study the vitamin D 25-hydroxylation in human subjects using cells from extrahepatic organs, from which biopsy retrieval is easier than from the liver. Dermal fibroblasts are frequently used to study different pathological conditions in human subjects and they are easy to come by. Dermal fibroblasts were shown to express two vitamin D 25-hydroxylases: CYP27A1 and CYP2R1. The expression pattern of CYP2R1 displayed considerable interindividual variation. The fibroblasts were also capable of measurable vitamin D 25-hydroxylation, which makes dermal fibroblasts a possible tool in studying vitamin D 25-hydroxylation in human subjects. Little is known about the regulation of expression and activity of the human vitamin D 25-hydroxylases. Therefore dermal fibroblasts – expressing CYP2R1 and CYP27A1 – and human prostate cancer LNCaP cells, that express CYP2R1 and CYP2J2, were treated with calcitriol and phenobarbital and efavirenz, two drugs that give rise to vitamin D deficiency. Treatment decreased the mRNA levels of CYP2R1 and CYP2J2 provided that the treated cells also expressed the necessary nuclear receptors. CYP27A1 did not respond to any of the treatments. The treatments also managed to decrease the 25-hydroxylating activity of the cells. The results show that vitamin D 25-hydroxylases can be regulated by both endogenous and xenobiotic compounds.

It is becoming increasingly clear that long-range control of gene expression is mediated through direct physical interactions between genes and regulatory elements, either intra- or interchromosomally. In addition to transcriptional initiation, formation of active chromatin hubs seem to be crucial for increased transcriptional efficiency as well as insulation from neighbouring heterochromatic environment. Regulatory factors apparently have an important role in organization of such functional modules in a development and differentiated- dependent fashion. The relevance of trans-acting factors in the ‘choice’ process of X-Chromosome Inactivation (XCI) was highlighted by our observations where CTCF was shown to occupy a homologous position on the active mouse and human Xist/XIST promoters and its binding affinity was altered in familial cases of opposite skewed X-inactivation patterns. The paradigm of genomic imprinting, i.e. the Igf2-H19 locus, manifests its imprinted states through the H19 Imprinting Control Region (ICR). The repression of the maternal Igf2 allele depends on the insulator properties of the H19 ICR when this interacts with CTCF. The studies here detected a novel kind of CTCF-dependent tightly closed pocket- like higher order structure exclusively on maternal allele which was found to be essential for imprinted Igf2 expression as well as maintenance of precise epigenetic marks at various Differentially Methylated Regions (DMRs) across this locus. Despite the highly condensed state of the mitotic chromosome, the insulator protein CTCF was found to constitutively occupy its known target sites. Furthermore, pivotal CTCF-dependent long-range regulatory loops within Igf2-H19 locus were found to survive mitotic compaction and such mechanisms might serve as a novel kind of epigenetic memory to minimize transcriptional chaos and to reset proper expression domains in the daughter cells as soon as cells exit mitosis. Our observations also suggest that the epigenetic reprogramming of H19 ICR during spermatogenesis is initiated by a CTCF-dependent recruitment of chromatin remodeling factor Lsh to the H19 ICR followed by completion of the imprint acquisition process by a replacement of CTCF with its closely related paralogue termed BORIS. Overall, this thesis unravels the novel roles for CTCF as an architectural factor in the organization of higher order chromatin conformations and transcriptional regulation.

Accelerated global warming plus an increasing need for energy is an equation not easily solved, thus new forms of sustainable energy production are urgently requested. In this context hydrogen production based on a cyanobacterial system offers an environmentally friendly alternative for energy capture and conversion. Cyanobacteria can produce hydrogen gas from sun light and water through the combination of photosystems and hydrogenases, and are suitable to cultivate in large scale. In the present thesis the maturation process of [NiFe]-hydrogenases is investigated with special focus on transcription of the accessory genes encoding proteins needed for assembly of the large and possibly also for the small hydrogenase subunit. The cyanobacteria used are two N2-fixing, filamentous, heterocystous strains; Nostoc sp. strain PCC 7120 and Nostoc punctiforme PCC 73102. For a biotechnological exploration of hydrogen production tools for regulatory purposes are important. The transcription factor CalA (cyanobacterial AbrB like) (Alr0946 in the genome) in Nostoc sp. strain PCC 7120 was found to be involved in hydrogen metabolism by regulating the transcription of the maturation protein HypC. Further the bidirectional hydrogenase activity was down-regulated in the presence of elevated levels of CalA, a result important to take into account when optimizing cyanobacteria for hydrogen production. CalA regulates at least 25 proteins in Nostoc sp. strain PCC 7120 and one of the down-regulated proteins was superoxide dismutase, FeSOD. The characterization of FeSOD shows that it has a specific and important function in the oxidative stress tolerance of Nostoc sp. stain PCC 7120. Since CalA is involved in regulation of both the hydrogen metabolism as well as stress responses these findings indicate that Alr0946 is an important transcription factor in Nostoc sp. strain PCC 7120 active on a global level in the cell. This thesis adds more knowledge concerning maturation and regulation of cyanobacterial hydrogenases which might be useful for future large scale hydrogen.

Gene activity is regulated at two separate layers. Through structural and chemical properties of DNA – the primary layer of encoding – local signatures may enable, or disable, the binding of proteins or complexes of them with regulatory potential to the DNA. At a higher level – the structural layer of encoding – gene activity is regulated through the properties of higher order DNA structure, chromatin, and chromosome organization. Cells with abnormal chromosome compaction or organization, e.g. cancer cells, may thus have perturbed regulatory activities resulting in abnormal gene activity. Hence, there is a great need to decode the transcriptional regulation encoded in both layers to further our understanding of the factors that control activity and life of a cell and, ultimately, an organism. Modern genome-wide studies with those aims rely on data-intense experiments requiring sophisticated computational and statistical methods for data handling and analyses. This thesis describes recent advances of analyzing experimental data from quantitative biological studies to decipher the structural layer of encoding in human cells. Adopting an integrative approach when possible, combining multiple sources of data, allowed us to study the influences of chromatin (Papers I and II) and chromosomal aberrations (Paper IV) on transcription. Combining chromatin data with chromosomal aberration data allowed us to identify putative driver oncogenes and tumor-suppressor genes in cancer (Paper IV). Bayesian approaches enabling the incorporation of background information in the models and the adaptability of such models to data have been very useful. Their usages yielded accurate and narrow detection of chromosomal breakpoints in cancer (Papers III and IV) and reliable positioning of nucleosomes and their dynamics during transcriptional regulation at functionally relevant regulatory elements (Paper II). Using massively parallel sequencing data, we explored the chromatin landscapes of human cells (Papers I and II) and concluded that there is a preferential and evolutionary conserved positioning at internal exons nearly unaffected by the transcriptional level. We also observed a strong association between certain histone modifications and the inclusion or exclusion of an exon in the mature gene transcript, suggesting a functional role in splicing.

The precise regulation of Major Histocompatibility class II (MHC-II) genes plays an important role in the control of the adaptive immune response. MHC-II genes are expressed constitutively in only a few cell types, but their expression can be induced by the inflammatory response cytokine interferon gamma (INF-γ). The regulation of MHC-II is controlled by a Master Regulator, the class II transactivator (CIITA). Multiple studies have shown that CIITA regulated expression of MHC-II is controlled and induced by INF-γ. It has been also shown that a functional CIITA gene is necessary for the expression of MHC-II genes. CIITA is thus a general regulator of both constitutive and inducible MHC-II expression. Although much is known about the transcription factors necessary for CIITA expression, there is little information as to the epigenetic modifications and the requisite enzymes needed to provide these transcription factors access to DNA. Previous studies in the Greer lab have shown that increased levels of acetylation of histones H3 upon INF-γ stimulation, as does tri-methylation of H3K4 upon prolonged cytokine stimulation. Similar observations were made at early time points post IFN-γ stimulation, where there is an instantaneous increase in the levels of H3K18ac and H3K4me3. In contrast to this, the levels of silencing modifications begin to drop with in the first 20 minutes of IFN-γ stimulation. The binding of STAT1 reaches its peak at about 60 minutes and the first transcripts for the protein start to appear as early as 40 minutes post the cytokines stimulation. Our study is the first to link the rapidly occurring epigenetic changes at the CIITA promoter pIV to EZH2