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DNA methylation is a key epigenetic modification to regulate gene expression in mammalian cells. Abnormal DNA methylation in gene promoters is common across human cancer types. DNMT3B is the main de novo methyltransferase enhanced in several primary tumors. How de novo methylation is established in genes related to cancer is poorly understood. CpG islands (CGIs), common sequences, and transcription factors (TFs) that interact with DNMT3B have been associated with abnormal de novo methylation. We initially identified cis elements associated with DNA methylation to investigate the contribution of DNMT3B overexpression to the deregulation of its possible target genes in an epithelial cell model. In a set of downregulated genes (n = 146) from HaCaT cells with DNMT3B overexpression, we found CGI, common sequences, and TFs Binding Sites that interact with DNMT3B (we called them P-down-3B). PPL1, VAV3, IRF1, and BRAF are P-down-3B genes that are downregulated and increased their methylation in DNMT3B presence. Together these findings suggest that methylated promoters aberrantly have some cis elements that could conduce de novo methylation by DNMT3B.

Chronic hypoxia is a major contributor to Chronic Kidney Disease (CKD) after Acute Kidney Injury (AKI). However, the temporal relation between the acute insult and maladaptive renal response to hypoxia remains unclear. In this study, we analyzed the time-course of renal hemodynamics, oxidative stress, inflammation, and fibrosis, as well as epigenetic modifications, with focus on HIF1α/VEGF signaling, in the AKI to CKD transition. Sham-operated, right nephrectomy (UNx), and UNx plus renal ischemia (IR + UNx) groups of rats were included and studied at 1, 2, 3, or 4 months. The IR + UNx group developed CKD characterized by progressive proteinuria, renal dysfunction, tubular proliferation, and fibrosis. At first month post-ischemia, there was a twofold significant increase in oxidative stress and reduction in global DNA methylation that was maintained throughout the study. Hif1α and Vegfa expression were depressed in the first and second-months post-ischemia, and then Hif1α but not Vegfa expression was recovered. Interestingly, hypermethylation of the Vegfa promoter gene at the HIF1α binding site was found, since early stages of the CKD progression. Our findings suggest that renal hypoperfusion, inefficient hypoxic response, increased oxidative stress, DNA hypomethylation, and, Vegfa promoter gene hypermethylation at HIF1α binding site, are early determinants of AKI-to-CKD transition.

Thousands of long noncoding RNAs (lncRNAs) are actively transcribed in mammalian genomes. This class of RNAs has important regulatory functions in a broad range of cellular processes and diseases. Numerous lncRNAs have been demonstrated to mediate gene regulation through RNA-based mechanisms. Simultaneously, non-functional lncRNA transcripts derived from the activity of lncRNA loci have been identified, which underpin the notion that a considerable fraction of lncRNA loci exert regulatory functions through mechanisms associated with the production or the activity of lncRNA loci beyond the synthesized transcripts. We particularly distinguish two main RNA-independent components associated with regulatory effects; the act of transcription and the activity of DNA regulatory elements. We describe the experimental approaches to distinguish and understand the functional mechanisms derived from lncRNA loci. These scenarios reveal emerging mechanisms important to understanding the lncRNA implications in genome biology.

CRISPR-Cas9 is a method for genome editing that can be used efficiently for in vivo applications; the basic implementation of this method is used to generate genome site-directed sequence eliminations. Here we describe a protocol for genome editing using CRISPR-Cas9 in zebrafish (Danio rerio) one-cell embryos.

Anastrepha ludens is a major pest of fruits including citrus and mangoes in Mexico and Central America with major economic and social impacts. Despite its importance, our knowledge on its embryonic development is scarce. Here, we report the first cytological study of embryonic development in A. ludens and provide a transcriptional landscape during key embryonic stages. We established 17 stages of A. ludens embryogenesis that closely resemble the morphological events observed in Drosophila. In addition to the extended duration of embryonic development, we observed notable differences including yolk extrusion at both poles of the embryo, distinct nuclear division waves in the syncytial blastoderm and a heterochronic change during the involution of the head. Characterization of the transcriptional dynamics during syncytial blastoderm, cellular blastoderm and gastrulation, showed that approximately 9000 different transcripts are present at each stage. Even though we identified most of the transcripts with a role during embryonic development present in Drosophila, including sex determination genes, a number of transcripts were absent not only in A. ludens but in other tephritids such as Ceratitis capitata and Bactrocera dorsalis. Intriguingly, some A. ludens embryo transcripts encode proteins present in other organisms but not in other flies. Furthermore, we developed an RNA in situ hybridization protocol that allowed us to obtain the expression patterns of genes whose functions are important in establishing the embryonic body pattern. Our results revealed novel tephritid-specific features during A. ludens embryonic development and open new avenues for strategies aiming to control this important pest.

Chromosomes are organized into high-frequency chromatin interaction domains called topologically associating domains (TADs), which are separated from each other by domain boundaries. The molecular mechanisms responsible for TAD formation are not yet fully understood. In Drosophila, it has been proposed that transcription is fundamental for TAD organization while the participation of genetic sequences bound by architectural proteins (APs) remains controversial. Here, we investigate the contribution of domain boundaries to TAD organization and the regulation of gene expression at the Notch gene locus in Drosophila. We find that deletion of domain boundaries results in TAD fusion and long-range topological defects that are accompanied by loss of APs and RNA Pol II chromatin binding as well as defects in transcription. Together, our results provide compelling evidence of the contribution of discrete genetic sequences bound by APs and RNA Pol II in the partition of the genome into TADs and in the regulation of gene expression in Drosophila., In Drosophila, transcription is thought to be required for TAD formation, while the role of architectural proteins remains controversial. Here, the authors find that deletion of domain boundaries at the fly Notch locus results in TAD fusion and long-range topological defects, loss of architectural protein and RNA Pol II chromatin binding, and transcription defects.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most intractable and devastating malignant tumors. Epigenetic modifications such as DNA methylation and histone modification regulate tumor initiation and progression. However, the contribution of histone variants in PDAC is unknown. Here, we demonstrated that the histone variant H2A.Z is highly expressed in PDAC cell lines and PDAC patients and that its overexpression correlates with poor prognosis. Moreover, all three H2A.Z isoforms (H2A.Z.1, H2A.Z.2.1, and H2A.Z.2.2) are highly expressed in PDAC cell lines and PDAC patients. Knockdown of these H2A.Z isoforms in PDAC cell lines induces a senescent phenotype, cell cycle arrest in phase G2/M, increased expression of cyclin-dependent kinase inhibitor CDKN2A/p16, SA-β-galactosidase activity and interleukin 8 production. Transcriptome analysis of H2A.Z-depleted PDAC cells showed altered gene expression in fatty acid biosynthesis pathways and those that regulate cell cycle and DNA damage repair. Importantly, depletion of H2A.Z isoforms reduces the tumor size in a mouse xenograft model in vivo and sensitizes PDAC cells to gemcitabine. Overexpression of H2A.Z.1 and H2A.Z.2.1 more than H2A.Z.2.2 partially restores the oncogenic phenotype. Therefore, our data suggest that overexpression of H2A.Z isoforms enables cells to overcome the oncoprotective barrier associated with senescence, favoring PDAC tumor grow and chemoresistance. These results make H2A.Z a potential candidate as a diagnostic biomarker and therapeutic target for PDAC.

In mammals, DNA methylation is a crucial epigenetic modification with key functions during development. Cellular processes that are regulated by DNA methylation comprise X chromosome inactivation, gene imprinting, genomic stability and transcriptional regulation. Generally, the methylation status of the majority of target sites is reliably propagated during mitosis. However, advances in genome-wide DNA methylation analysis at base-resolution have discovered a substantial amount of differential DNA methylation between normal cells of different tissue-origin. Moreover, aberrant DNA methylation changes are linked with a significant number of human diseases, particularly with cancer. Sites of differential and aberrant DNA methylation include regulatory DNA sequences, such as CpG islands in promoters and distal cis-regulatory elements, like enhancers. In this review, we will discuss novel aspects of DNA methylation dynamics, during normal development and in association with diseases.

Sex determination in vertebrates depends on the expression of a conserved network of genes. Sea turtles such as Lepidochelys olivacea have temperature-dependent sex determination. The present work analyses some of the epigenetic processes involved in this. We describe sexual dimorphism in global DNA methylation patterns between ovaries and testes of L. olivacea and show that the differences may arise from a combination of DNA methylation and demethylation events that occur during sex determination. Irrespective of incubation temperature, 5-hydroxymethylcytosine was abundant in the bipotential gonad; however, following sex determination, this modification was no longer found in pre-Sertoli cells in the testes. These changes correlate with the establishment of the sexually dimorphic DNA methylation patterns, down regulation of Sox9 gene expression in ovaries and irreversible gonadal commitment towards a male or female differentiation pathway. Thus, DNA methylation changes may be necessary for the stabilization of the gene expression networks that drive the differentiation of the bipotential gonad to form either an ovary or a testis in L. olivacea and probably among other species that manifest temperature-dependent sex determination.

Over the years, the study of gene function during development involved the implementation of sophisticated transgenic strategies to visualize how organisms change during their lifetime. These strategies are diverse and extremely useful and allowed the discovery of some of the fundamental mechanisms governing organism's development. Such strategies can be time-consuming, in some cases expensive, and require complex infrastructure. With the advent of the genome editing CRISPR-Cas9 RNA-guided DNA endonuclease system a tremendous progress has been achieved in manipulating diverse organisms and cell types. In recent years this system has contributed importantly to the design of novel experimental strategies to further understand developmental processes, to generate genetically modified animal models, and develop disease models. Here we highlight examples in which the genome editing CRISPR-Cas9 system has been employed to understand the mechanisms controlling embryonic development and disease.

Lamin A is part of a complex structural meshwork located beneath the nuclear envelope and is involved in both structural support and the regulation of gene expression. Lamin A is initially expressed as prelamin A, which contains an extended carboxyl terminus that undergoes a series of post-translational modifications and subsequent cleavage by the endopeptidase ZMPSTE24 to generate lamin A. To facilitate investigations of the role of this cleavage in normal and disease states, we developed a monoclonal antibody (PL-1C7) that specifically recognizes prelamin A at the intact ZMPSTE24 cleavage site, ensuring prelamin A detection exclusively. Importantly, PL-1C7 can be used to determine prelamin A localization and accumulation in cells where lamin A is highly expressed without the use of exogenous fusion proteins. Our results show that unlike mature lamin A, prelamin A accumulates as discrete and localized foci at the nuclear periphery. Furthermore, whereas treatment with farnesylation inhibitors of cells overexpressing a GFP-prelamin A fusion protein results in the formation of large nucleoplasmic clumps, these aggregates are not observed upon similar treatment of cells expressing endogenous prelamin A or in cells lacking ZMPSTE24 expression and/or activity. Finally, we show that specific laminopathy-associated mutations exhibit both positive and negative effects on prelamin A accumulation, indicating that these mutations affect prelamin A processing efficiency in different manners.

Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish.

Gene promoters are frequently insufficient to drive the spatiotemporal patterns of gene expression during cell differentiation and organism development. Enhancers convey these properties through diverse mechanisms, including long-distance interactions with target promoters via their association with specific transcription factors. Despite unprecedented progress in the knowledge of enhancer mechanisms of action, there are still many unanswered questions. In particular, the contribution of an enhancer's local chromatin configuration to its mechanism of action is not completely understood. Here we describe a novel regulatory element, the Upstream Enhancer Element (UEE), which modulates the activity of the chicken α-globin 3′ enhancer by regulating its chromatin structure, specifically by positioning a nucleosome upstream of the core enhancer. This element binds nuclear factors and confers a more restricted activation on the α-globin 3′ enhancer, suggesting a progressive/rheostatic model for enhancer activity. Our results suggest that the UEE activity contributes to the positioning of a nucleosome that is necessary for the α-globin 3′ enhancer activation.

The estrogen receptor alpha (ERα) is a ligand-activated transcription factor that possesses two activating domains designated AF-1 and AF-2 that mediate its transcriptional activity. The role of AF-2 is to recruit coregulator protein complexes capable of modifying chromatin condensation status. In contrast, the mechanism responsible for the ligand-independent AF-1 activity and for its synergistic functional interaction with AF-2 is unclear. In this study, we have identified the protein Na+/H+ Exchanger RegulatoryFactor 2 (NHERF2) as an ERα-associated coactivator that interacts predominantly with the AF-1 domain of the nuclear receptor. Overexpression of NHERF2 in breast cancer MCF7 cells produced an increase in ERα transactivation. Interestingly, the presence of SRC-1 in NHERF2 stably overexpressing MCF7 cells produced a synergistic increase in ERα activity. We show further that NHERF2 interacts with ERα and SRC-1 in the promoter region of ERα target genes. The binding of NHERF2 to ERα in MCF7 cells increased cell proliferation and the ability of MCF7 cells to form tumors in a mouse model. We analyzed the expression of NHERF2 in breast cancer tumors finding a 2- to 17-fold increase in its mRNA levels in 50% of the tumor samples compared to normal breast tissue. These results indicate that NHERF2 is a coactivator of ERα that may participate in the development of estrogen-dependent breast cancer tumors.

Long non-coding RNAs (lncRNAs) were recently shown to regulate chromatin remodelling activities. Their function in regulating gene expression switching during specific developmental stages is poorly understood. Here we describe a nuclear, non-coding transcript responsive for the stage-specific activation of the chicken adult α(D) globin gene. This non-coding transcript, named α-globin transcript long non-coding RNA (lncRNA-αGT) is transcriptionally upregulated in late stages of chicken development, when active chromatin marks the adult α(D) gene promoter. Accordingly, the lncRNA-αGT promoter drives erythroid-specific transcription. Furthermore, loss of function experiments showed that lncRNA-αGT is required for full activation of the α(D) adult gene and maintenance of transcriptionally active chromatin. These findings uncovered lncRNA-αGT as an important part of the switching from embryonic to adult α-globin gene expression, and suggest a function of lncRNA-αGT in contributing to the maintenance of adult α-globin gene expression by promoting an active chromatin structure.

The pathological characteristic of cirrhosis is scarring which results in a structurally distorted and dysfunctional liver. Previously, we demonstrated that Col1a1 and Pparg genes are deregulated in CCl4 -induced cirrhosis but their normal expression levels are recovered upon treatment with IFC-305, an adenosine derivative. We observed that adenosine was able to modulate S-adenosylmethionine-dependent trans-methylation reactions, and recently, we found that IFC-305 modulates HDAC3 expression. Here, we investigated whether epigenetic mechanisms, involving DNA methylation processes and histone acetylation, could explain the re-establishment of gene expression mediated by IFC-305 in cirrhosis. Therefore, Wistar rats were CCl4 treated and a sub-group received IFC-305 to reverse fibrosis. Global changes in DNA methylation, 5-hydroxymethylation, and histone H4 acetylation were observed after treatment with IFC-305. In particular, during cirrhosis, the Pparg gene promoter is depleted of histone H4 acetylation, whereas IFC-305 administration restores normal histone acetylation levels which correlates with an increase of Pparg transcript and protein levels. In contrast, the promoter of Col1a1 gene is hypomethylated during cirrhosis but gains DNA methylation upon treatment with IFC-305 which correlates with a reduction of Col1a1 transcript and protein levels. Our results suggest a model in which cirrhosis results in a general loss of permissive chromatin histone marks which triggers the repression of the Pparg gene and the upregulation of the Col1a1 gene. Treatment with IFC-305 restores epigenetic modifications globally and specifically at the promoters of Pparg and Col1a1 genes. These results reveal one of the mechanisms of action of IFC-305 and suggest a possible therapeutic function in cirrhosis. J. Cell. Biochem. 119: 401-413, 2018. © 2017 Wiley Periodicals, Inc.

Cells must be able to respond rapidly and precisely not only to changes in their external environment but also to developmental and differentiation cues to determine when to divide, die, or acquire a particular cell fate. Signal transduction pathways are responsible for the integration and interpretation of most of such signals into specific transcriptional states. Those states are achieved by the modulation of chromatin structure that activates or represses transcription at particular loci. Although a large variety of signal transduction pathways have already been described, much less is known about the crosstalk between signal transduction and its consequent changes in chromatin structure and, therefore, gene expression. Here we present some examples of the relationship between chromatin-associated proteins and important signal transduction pathways during critical processes like development, differentiation, and disease. There is a great diversity of epigenetic mechanisms that have unexpected interactions with signaling pathways to establish transcriptional programs. Moreover, there are also particular cases where signaling pathways directly affect important components of the epigenetic machinery. Based on such examples, we further propose future research directions linking cell signaling and epigenetics. It is foreseeable that analyzing the relationship between cell signaling and epigenetics will be a huge area for future development that will help us understand the complex process by which a cell is able to induce transcriptional changes in response to external and internal signals.

The p53 tumor suppressor is one of the most studied molecules in cancer research. Despite the fact that there is a detailed understanding involving multiple aspects of the p53-associated biology, many aspects of its transcriptional regulation are still not well clarified. Limited information is available on how the p53 gene is transcriptional and epigenetically regulated. The p53 gene expression is tightly controlled through a variety of transcription factors, miRNAs, its anti-sense RNA Wrap53, the insulator protein CTCF and very likely by other genetic and epigenetic mechanisms. It's the intent of this article to review in depth important aspects concerning the transcriptional regulation of the p53 gene and perhaps serve as a stepping-stone to begin a conceptual change on how future p53 research can be approached.

Small neutral amino acid transporter 2 (SNAT2) is the most abundant and ubiquitous transporter for zwitterionic short-chain amino acids. The activity of this amino acid transporter is stimulated in vivo or in vitro by glucagon or cAMP analogs. However, it is not known whether the increase in activity at the protein level is due to an increase in SNAT2 gene transcription. Thus, the aim of the present work was to study whether cAMP was able to stimulate SNAT2 gene expression and to localize and characterize the presence of cAMP response elements (CRE) in the promoter that controls the expression of the rat SNAT2 gene. We found that consumption of a high-protein diet that increased serum glucagon concentration or the administration of glucagon or incubation of hepatocytes with forskolin increased the SNAT2 mRNA level. We then isolated the 5′ regulatory region of the SNAT2 gene and determined that the transcriptional start site was located 970 bp upstream of the translation start codon. We identified two potential CRE sites located at −354 and −48 bp. Our results, using deletion analysis of the 5′ regulatory region of the SNAT2 gene, revealed that the CRE site located at −48 bp was fully responsible for SNAT2 regulation by cAMP. This evidence was strongly supported by mutation of the CRE site and EMSA and ChIP analysis. Alignment of rat, mouse, and human sequences revealed that this CRE site is highly conserved among species, indicating its essential role in the regulation of SNAT2 gene expression.

7 páginas, 6 figuras, 1 tabla.-- et al., PMCID: PMC3196567, Many genomic alterations associated with human diseases localize in noncoding regulatory elements located far from the promoters they regulate, making it challenging to link noncoding mutations or risk-associated variants with target genes. The range of action of a given set of enhancers is thought to be defined by insulator elements bound by the 11 11 11 11 zinc-finger nuclear factor CCCTC-binding protein (CTCF). Here we analyzed the genomic distribution of CTCF in various human, mouse and chicken cell types, demonstrating the existence of evolutionarily conserved CTCF-bound sites beyond mammals. These sites preferentially flank transcription factor–encoding genes, often associated with human diseases, and function as enhancer blockers in vivo, suggesting that they act as evolutionarily invariant gene boundaries. We then applied this concept to predict and functionally demonstrate that the polymorphic variants associated with multiple sclerosis located within the EVI5 gene impinge on the adjacent gene GFI1., This research was supported by the following grants: BFU2007-60042/BMC, BFU2010-14839, Petri PET2007_0158, CONSOLIDER CSD2007-00008 (Spanish Ministerio de Ciencia e Innovación (MICINN)) and Proyecto de Excelencia CVI-3488 (Junta de Andalucía) to J.L.G.-S.; BFU2009-07044 (MICINN) and Proyecto de Excelencia CVI 2658 (Junta de Andalucía) to F.C.; FIS PI081636 (ISCIII) to F.M.; PN-SAF2009-11491 (MICINN) and Proyecto de Excelencia P07-CVI-02551 (Junta de Andalucía) to A.A.; BFU2008-00838, CONSOLIDER CSD2007-00008 (MICINN), Regional Government of Madrid (CAM S-SAL-0190-2006) and the Pro-CNIC Foundation to M.M.; BFU2006-12185 and BIO2009-12697 (MICINN) to L.M.; Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México (IN209403, IN214407 and IN203811) and Consejo Nacional de Ciencia y Tecnología, México (CONACyT: 42653-Q, 58767 and 128464) to F.R.-T.; Intramural Research Program of the US NCBI (NIH) to I.O. and BIO2006-03380, CONSOLIDER CSD2007-00050 (MICINN) and RETICS RD07/0067/0012 (Spanish MICINN) to R.G. L.M. thanks A. Fernández for technical assistance and L. Barrios for statistical analysis. F.R.-T. thanks G.G. Avendaño for technical assistance.

Pluripotent embryonic stem (ES) cells are specialized cells with a dynamic chromatin structure, which is intimately connected with their pluripotency and physiology. In recent years somatic cells have been reprogrammed to a pluripotent state through over-expression of a defined set of transcription factors. These cells, known as induced pluripotent stem (iPS) cells, recapitulate ES cell properties and can be differentiated to apparently all cell lineages, making iPS cells a suitable replacement for ES cells in future regenerative medicine. Chromatin modifiers play a key function in establishing and maintaining pluripotency, therefore, elucidating the mechanisms controlling chromatin structure in both ES and iPS cells is of utmost importance to understanding their properties and harnessing their therapeutic potential. In this review, we discuss recent studies that provide a genome-wide view of the chromatin structure signature in ES cells and iPS cells and that highlight the central role of histone modifiers and chromatin remodelers in pluripotency maintenance and induction.

Genome organization into transcriptionally active domains denotes one of the first levels of gene expression regulation. Although the chromatin domain concept is generally accepted, only little is known on how domain organization impacts the regulation of differential gene expression. Insulators might hold answers to address this issue as they delimit and organize chromatin domains. We have previously identified a CTCF-dependent insulator with enhancer-blocking activity embedded in the 5' non-coding region of the chicken α-globin domain. Here, we demonstrate that this element, called the αEHS-1.4 insulator, protects a transgene against chromosomal position effects in stably transfected cell lines and transgenic mice. We found that this insulator can create a regulated chromatin environment that coincides with the onset of adult α-globin gene expression. Furthermore, such activity is in part dependent on the in vivo regulated occupancy of CTCF at the αEHS-1.4 element. Insulator function is also regulated by CTCF poly(ADP-ribosyl)ation. Our results suggest that the αEHS-1.4 insulator contributes in organizing the chromatin structure of the α-globin gene domain and prevents activation of adult α-globin gene expression at the erythroblast stage via CTCF.

CTCF is a highly conserved zinc-finger DNA-binding protein that mediates interactions between distant sequences in the genome. As a consequence, CTCF regulates enhancer-promoter interactions and contributes to the three-dimensional organization of the genome. Recent studies indicate that CTCF is developmentally regulated, suggesting that it plays a role in cell type-specific genome organization. Here, we review these studies and discuss how CTCF functions during the development of various cell and tissue types, ranging from embryonic stem cells and gametes, to neural, muscle and cardiac cells. We propose that the lineage-specific control of CTCF levels, and its partnership with lineage-specific transcription factors, allows for the control of cell type-specific gene expression via chromatin looping.

Meiosis is a key cellular and molecular process for sexual reproduction contributing to the genetic variability of organisms. This process takes place after DNA replication and consists in a double cellular division, giving rise to four haploid daughter cells or gametes. Meiotic recombination between homologous chromosomes, in the meiotic prophase I, is mediated by a tripartite structure named Synaptonemal Complex (SC). The SC is a peptidic scaffold in which the chromatin of homologous chromosomes is organized during the pachytene stage, holding chromosomes together until the meiotic recombination and genetic exchange have taken place. The role of chromatin structure in formation of the SC and the meiotic recombination at meiotic prophase I remain largely unknown. In this review we address the epigenome contribution to the SC formation at meiotic prophase I, with particular attention on the chromatin structure modifications occurring during the sub-stages of meiotic prophase I.

The interdependency between genetic and epigenetic regulatory processes renders cancer therapeutics and translational clinic possible, even though they remain difficult tasks considering all the evidence supporting a central role of progenitor-stem cells as a causative source of cellular transformation. In contrast to genetic alterations, epigenetic processes are potentially reversible allowing a better action of complementary therapeutic compounds. Here I would first describe the plethora of interconnected epigenetic processes and targets to then discuss several therapeutic strategies on the basis of different compounds. I conclude that the advent of new and specific epigenetic target drugs will certainly contribute to better treatments and to the development of predictive protocols in a next future.

Long-distance regulatory elements and local chromatin structure are critical for proper regulation of gene expression. Here we characterize the chromatin conformation of the chicken alpha-globin silencer-enhancer elements located 3' of the domain. We found a characteristic and erythrocyte-specific structure between the previously defined silencer and the enhancer, defined by two nuclease hypersensitive sites, which appear when the enhancer is active during erythroid differentiation. Fine mapping of these sites demonstrates the absence of a positioned nucleosome and the association of GATA-1. Functional analyses of episomal vectors, as well as stably integrated constructs, revealed that GATA-1 plays a major role in defining both the chromatin structure and the enhancer activity. We detected a progressive enrichment of histone acetylation on critical enhancer nuclear factor binding sites, in correlation with the formation of an apparent nucleosome-free region. On the basis of these results, we propose that the local chromatin structure of the chicken alpha-globin enhancer plays a central role in its capacity to differentially regulate alpha-globin gene expression during erythroid differentiation and development.

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC.

During organism development, a diversity of cell types emerges with disparate, yet stable profiles of gene expression with distinctive cellular functions. In addition to gene promoters, the genome contains enhancer regulatory sequences, which are implicated in cellular specialization by facilitating cell-type and tissue-specific gene expression. Enhancers are DNA binding elements characterized by highly sophisticated and various mechanisms of action allowing for the specific interaction of general and tissue-specific transcription factors (TFs). However, eukaryotic organisms package their genetic material into chromatin, generating a physical barrier for TFs to interact with their cognate sequences. The ability of TFs to bind DNA regulatory elements is also modulated by changes in the chromatin structure, including histone modifications, histone variants, ATP-dependent chromatin remodeling, and the methylation status of DNA. Furthermore, it has recently been revealed that enhancer sequences are also transcribed into a set of enhancer RNAs with regulatory potential. These interdependent processes act in the context of a complex network of chromatin interactions, which together contributes to a renewed vision of how gene activation is coordinated in a cell-type-dependent manner. In this review, we describe the interplay between genetic and epigenetic aspects associated with enhancers and discuss their possible roles on enhancer function.

Genetic and epigenetic regulations are essential mechanisms that ensure proper early and subsequent mammalian programming of diverse cellular processes. These mechanisms affect transcriptional regulation, stem cell determination and cell cycle control, including senescence and aging. It is not surprising that perturbation of the exquisite balance between genetic and epigenetic regulation can lead to diverse diseases, including cancer. Histone covalent modifications and DNA methylation do not explain all epigenetic phenomena. We describe a previously unsuspected epigenetic factor and propose the incorporation of the 11-zinc finger CCCTC-binding factor, known as CTCF as a novel and multifunctional epigenetic regulator.

The tissue-specific chicken alpha-globin gene domain represents one of the paradigms, in terms of its constitutively open chromatin conformation and the location of several regulatory elements within the neighboring housekeeping gene. Here, we show that an 0.2-kb DNA fragment located approximately 4 kb upstream to the chicken alpha-globin gene cluster contains a binding site for the multifunctional protein factor CTCF and possesses silencer activity which depends on CTCF binding, as demonstrated by site-directed mutagenesis of the CTCF recognition sequence. CTCF was found to be associated with this recognition site in erythroid cells but not in lymphoid cells where the site is methylated. A functional promoter directing the transcription of the apparently housekeeping ggPRX gene was found 120 bp from the CTCF-dependent silencer. The data are discussed in terms of the hypothesis that the CTCF-dependent silencer stabilizes the level of ggPRX gene transcription in erythroid cells where the promoter of this gene may be influenced by positive cis-regulatory signals activating alpha-globin gene transcription.

Manipulation of the eukaryotic genome has contributed to the progress in our knowledge of multicellular organisms but has also ameliorated our experimental strategies. Biological questions can now be addressed with more efficiency and reproducibility. There are new and varied strategies for gene transfer and sequence manipulation with improved methodologies that facilitate the acquisition of results. Cellular systems and transgenic animals have demonstrated their invaluable benefits. In this review, I present an overview of the methods of gene transfer with particular attention to cultured cell lines and large-scale sequence vectors, like artificial chromosomes, with the possibility of their manipulation based on homologous recombination strategies. Alternative strategies of gene transfer, including retroviral vectors, are also described and the applications of such methods are discussed. Finally, several comments are made about the influence of chromatin structure on gene expression. Recent experimental data have shown that for convenient stable transgene expression, the influence of chromatin structure should be seriously taken into account. Novel chromatin regulatory and structural elements are proposed as an alternative for proper and sustained gene expression. These chromatin elements are facing a new era in transgenesis and we are probably beginning a new generation of gene and cancer therapy vectors.

Studying the chicken alpha-globin domain as a model system of gene regulation, we have previously identified contiguous silencer-enhancer elements located on the 3'-side of the domain. To better characterize the enhancer we performed a systematic functional analysis to define its expression influence range and the ubiquitous and stage-specific transcriptional regulators interacting with this control element. In contrast to previous reports, we found that, in addition to a core element that includes three GATA-1 binding sites, the enhancer incorporates a 120 base-pair DNA fragment where EKLF, NF-E2 and a fourth GATA-1 factor could interact. Functional experiments demonstrate that the enhancer activity over the adult alpha(D) promoter is differentially regulated. We found that the transcriptional factor Ying Yang 1 (YY1) binds to the 120 base-pair DNA fragment and its effect over the enhancer activity is GATA-1-dependent. In addition, we characterize a novel physical interaction between GATA-1 and YY1 that influences the enhancer function. Experiments using a histone deacetylation inhibitor indicate that, in pre-erythroblasts, the enhancer down-regulation could be influenced by a closed chromatin conformation. Our observations show that the originally defined enhancer possesses a more complex composition than previously assumed. We propose that its activity is modulated through differential nuclear factor interactions and chromatin modifications at distinct erythroid stages.

RNA polymerase II promoters in Plasmodium spp., like in most eukaryotes, have a bipartite structure. However, the identification of a functional TATA box located within the Plasmodium spp. core promoters has been difficult, mainly because of its high A + T content. Only few putative trans-acting elements have been identified in the malaria parasite genome such as a gene orthologous to the TATA box binding protein (PfTBP). In this study, we demonstrate that PfTBP is part of the DNA–protein complexes formed in the kahrp and gbp-130 gene promoter regions. Supershift and footprinting assays performed with a GST–PfTBP fusion protein showed that PfTBP associates with a consensus TATA box sequence located 81 base pairs upstream of the transcription start site in the kahrp promoter region and with a TATA box-like (TGTAA) sequence at position −186 of the gbp-130 gene promoter region. Chromatin immunoprecipitation assays confirmed that native PfTBP is able to associate in vivo with both TATA box elements. This is the first study that reports the identification of cis-acting sequences (TATAA and TGTAA) and their correspondingtrans-acting (PfTBP) factor in P. falciparum. © 2005 Elsevier B.V. All rights reserved.

The eukaryotic genome is partitioned into chromatin domains containing coding and intergenic regions. Insulators have been suggested to play a role in establishing and maintaining chromatin domains. Here we describe the identification and characterization of two separable enhancer blocking elements located in the 5' flanking region of the chicken alpha-globin domain, 11-16 kb upstream of the embryonic alpha-type pi gene in a DNA fragment harboring a MAR (matrix attachment region) element and three DNase I hypersensitive sites (HSs). The most upstream enhancer blocking element co-localizes with the MAR element and an erythroid-specific HS. The second enhancer blocking element roughly co-localizes with a constitutive HS. The third erythroid-specific HS present within the DNA fragment studied harbors a silencing, but not an enhancer blocking, activity. The 11 zinc-finger CCCTC-binding factor (CTCF), which plays an essential role in enhancer blocking activity in many previously characterized vertebrate insulators, is found to bind the two alpha-globin enhancer blocking elements. Detailed analysis has demonstrated that mutation of the CTCF binding site within the most upstream enhancer blocking element abolishes the enhancer blocking activity. The results are discussed with respect to special features of the tissue-specific alpha-globin gene domain located in a permanently open chromatin area.

Gene therapy has emerged from the idea of inserting a wild-type copy of a gene in order to restore the proper expression and function of a damaged gene. Initial efforts have focused on finding the proper vector and delivery method to introduce a corrected gene to the affected tissue or cell type. Even though these first attempts are clearly promising, several problems remain unsolved. A major problem is the influence of chromatin structure on transgene expression. To overcome chromatin-dependent repressive transgenic states, researchers have begun to use chromatin regulatory elements to drive transgene expression. Insulators or chromatin boundaries are able to protect a transgene against chromatin position effects at their genomic integration sites, and they are able to maintain transgene expression for long periods of time. Therefore, these elements may be very useful tools in gene therapy applications for ensuring high-level and stable expression of transgenes.

Silencing of retrovirus vectors poses a significant obstacle to genetic manipulation of stem cells and their use in gene therapy. We describe a mammalian silencer blocking assay using insulator elements positioned between retrovirus silencer elements and an LCRbeta-globin reporter transgene. In transgenic mice, we show that retrovirus silencers are blocked by the cHS4 insulator. Silencer blocking is independent of the CTCF binding site and is most effective when flanking the internal reporter transgene. These data distinguish silencer blocking activity by cHS4 from its enhancer blocking activity. Retrovirus vectors can be created at high titer with one but not two internal dimer cHS4 cores. cHS4 in the LTRs has no effect on expression in transduced F9 cells, suggesting that position effect blocking is not sufficient to escape silencing. The Drosophila insulators gypsy and Scs fail to block silencing in transgenic mice, but gypsy stimulates vector expression 2-fold when located in the LTRs of an infectious retrovirus. The silencer blocking assay complements existing insulator assays in mammalian cells, provides new insight into mechanisms of insulation and is a valuable tool to identify additional silencer blocking insulators that cooperate with cHS4 to improve stem cell retrovirus vector design.

The three-dimensional architecture of genomes provides new insights about genome organization and function, but many aspects remain unsolved at the local genomic scale. Here we investigate the regulation of two erythroid-specific loci, a folate receptor gene (FOLR1) and the β-globin gene cluster, which are separated by 16. kb of constitutive heterochromatin. We found that in early erythroid differentiation the FOLR1 gene presents a permissive chromatin configuration that allows its expression. Once the transition to the next differentiation state occurs, the heterochromatin spreads into the FOLR1 domain, concomitant with the dissociation of CTCF from a novel binding site, thereby resulting in irreversible silencing of the FOLR1 gene. We demonstrate that the sequences surrounding the CTCF-binding site possess classical insulator properties in vitro and in vivo. In contrast, the chicken cHS4 β-globin insulator present on the other side of the heterochromatic segment is in a constitutive open chromatin configuration, with CTCF constantly bound from the early stages of erythroid differentiation. Therefore, this study demonstrates that the 16. kb of constitutive heterochromatin contributes to silencing of the FOLR1 gene during erythroid differentiation., This work was supported by the DGAPA-PAPIIT, UNAM (IN209403, IN203811 and IN201114), and CONACyT (42653-Q, 128464 and 220503) to FR-T, and by a Ph.D. fellowship from CONACyT (CVU 207989) and Programa de Apoyo a los Estudios del Posgrado (PAEP), UNAM to EG-B. Additional support was provided by the PhD Graduate Program, “Doctorado en Ciencias Bioquímicas”, to the Instituto de Fisiología Celular and the Universidad Nacional Autónoma de México. JLG-S was funded by grants from Ministerio de Economía y Competitividad (BFU2013-41322-P) and the Andalusian Government (BIO-396). A postdoctoral grant from the Universidad Pablo de Olavide supported JJT.

The 1.2-kb DNA sequence element (5′HS4) at the 5′ end of the chicken β-globin locus has the two defining properties of an insulator: it prevents an “external” enhancer from acting on a promoter when placed between them (“enhancer blocking”) and acts as a barrier to chromosomal position effect (CPE) when it surrounds a stably integrated reporter. We previously reported that a single CTCF-binding site in 5′HS4 is necessary and sufficient for enhancer blocking. We show here that a 250-bp “core” element from within 5′HS4 is sufficient to confer protection against silencing of transgenes caused by CPE. Further dissection of the core reveals that 5′HS4 is a compound element in which it is possible to separate enhancer blocking and barrier activities. We demonstrate that full protection against CPE is conferred by mutant 5′HS4 sequences from which the CTCF-binding site has been deleted. In contrast, mutations of four other protein binding sites within 5′HS4 result in varying reductions in the ability to protect against CPE. We find that binding sites for CTCF are neither necessary nor sufficient for protection against CPE. Comparison of the properties of 5′HS4 with those of other CTCF-binding enhancer-blocking elements suggests that CPE protection is associated with maintenance of a high level of histone acetylation near the insulator, conferred by insulator binding-proteins other than CTCF.

Epigenetic regulation is understood as heritable changes in gene expression and genome function that can occur without affecting the DNA sequence. In its in vivo context DNA is coupled to a group of small basic proteins that together with the DNA form the chromatin. The organization and regulation of the chromatin alliance with multiple nuclear functions are inconceivable without genetic information. With the advance on the understanding of the chromatin organization of the eukaryotic genome, it has been clear that not only genetics but also epigenetics influence both normal human biology and diseases. As a consequence, the basic concepts and mechanisms of cancer need to be readdressed and viewed not only locally but also at the whole genome scale or even, in the three-dimensional context of the cell nucleus space. Such a vision has a larger impact than has been previously predicted, since phenomena like aging, senescence, the entail of nutrition, stem cell biology, and cancer are orchestrated by epigenetic and genetic processes. Here I describe the relevance and central role of genetic and epigenetic defects in cancer.

Alzheimer's disease (AD) is a complex disorder whose etiology is associated with environmental and genetic factors. Recently there have been several attempts to analyze the role of epigenetic alterations in the origin and progression of this neurodegenerative condition. To evaluate the potential participation of the methylation status of the genome that may contribute to AD progression, we have studied the levels and distribution of the 5-methylcytosine and 5-hydroxymethylcytosine in different brain regions at different ages. We analyzed and quantified the immunosignal of these two epigenetic marks in young versus old wild-type mice and in the triple-transgenic mouse model of AD (3xTg-AD). The results show a decline in global 5-methylcytosine mark over time in all studied brain regions concomitant with a significant and widespread increase in 5-hydroxymethylcytosine mark in the aged transgenic mice in contrast to the age-matched controls. These differences in the methylation pattern of brain DNA in the 3xTg-AD that accumulates along age indicates abnormal formation of permissive chromatin structure associated with the increase in AD-related markers.

Genomic loci composed of more than one gene are frequently subjected to differential gene expression, with the chicken α-globin domain being a clear example. In the present study we aim to understand the globin switching mechanisms responsible for the epigenetic silencing of the embryonic π gene and the transcriptional activation of the adult α(D) and α(A) genes at the genomic domain level. In early stages, we describe a physical contact between the embryonic π gene and the distal 3' enhancer that is lost later during development. We show that such a level of regulation is achieved through the establishment of a DNA hypermethylation sub-domain that includes the embryonic gene and the adjacent genomic sequences. The multifunctional CCCTCC-binding factor (CTCF), which is located upstream of the α(D) gene promoter, delimits this sub-domain and creates a transition between the inactive sub-domain and the active sub-domain, which includes the adult α(D) gene. In avian-transformed erythroblast HD3 cells that are induced to differentiate, we found active DNA demethylation of the adult α(D) promoter, coincident with the incorporation of 5-hydroxymethylcytosine (5hmC) and concomitant with adult gene transcriptional activation. These results suggest that autonomous silencing of the embryonic π gene is needed to facilitate an optimal topological conformation of the domain. This model proposes that CTCF is contributing to a specific chromatin configuration that is necessary for differential α-globin gene expression during development.

To understand the mechanisms regulating the tissue non-specific alkaline phosphatase (TNAP) activity during development, we characterized cis-transcriptional regulatory elements. In embryonic cells and tissues, TNAP expression was driven preferentially by the exon 1A (E1A) promoter, one of the two promoters previously defined. Transcriptional activity of E1A promoter was up-regulated by retinoic acid (RA) through a putative RA-responsive element. Transgenic mice analysis with lacZ reporter constructs revealed negative regulatory elements within 8.5 kb of E1A promoter. Promoter sequences of endogenous TNAP in non-expressing tissues and those carried by the 8.5 kb-lacZ transgene were found to be highly methylated. A 1 kb fragment of E1A promoter increased the methylation level of lacZ and promoter sequences. The role of RA and DNA methylation in defining the embryonic expression pattern of TNAP is discussed.

One of the most outstanding nuclear factors, which has chromatin insulator and transcriptional properties and also contribute to genomic organization, is the zinc-finger protein CCCTC-binding factor (CTCF). Among its multiple functions, a growing amount of evidence implicates CTCF in the epigenetic regulation of genes responsible for the control of the cell cycle, and its mis-regulation can lead to aberrant epigenetic silencing of genes involved in cancer development. Detailed studies are now revealing that CTCF can serve as a barrier against the spread of DNA methylation and histone repressive marks over promoter regions of tumor suppressor genes. Moreover, new evidences points out to the capacity of CTCF to be covalently modified, in particular, through poly(ADP-ribosyl)ation with regulatory consequences. An unexplored aspect of CTCF is its intergenic and intragenic distribution in certain loci. Such distribution seems to facilitate the formation of an optimal chromatin structure and the recruitment of chromatin remodelers with the possible incorporation of RNA polymerase II. Therefore, in the context of tumor suppressor genes and cancer development, CTCF appears to play a relevant role by incorporating a combination of mechanisms involved in the protection against epigenetic silencing components and the maintenance of optimal higher-order organization of the corresponding loci.

Enhancer activities have been observed in DNA fragments up to 1.36 kb long located on the 3'-side of the cluster of the three alpha-type globin-encoding genes in duck [Kretsovali et al., C.R. Acad. Sci. Paris 307 (1988) 563-568] and chicken [Knezetic and Felsenfeld, Mol. Cell. Biol. 9 (1989) 893-901]. We report here the identification of a chicken silencer element placed upstream from the three GATA-1 sites which constitute the core enhancer element in both species. This silencer element can autonomously reduce the activity of promoters for thymidine kinase and alpha D globin. Band shifts and DNase I footprinting experiments using nuclear extracts from thermosensitive avian erythroblastosis virus-transformed chicken erythroblasts led to the delineation of three sites for DNA-binding proteins within the silencer element.

Embryonic neurogenesis is controlled by the activation of specific genetic programs. In the hypothalamus, neuronal thyrotropin-releasing hormone (TRH) populations control important physiological process, including energy homeostasis and autonomic function; however, the genetic program leading to the TRH expression is poorly understood. Here, we show that the Klf4 gene, encoding the transcription factor Kruppel-like factor 4 (Klf4), was expressed in the rat hypothalamus during development and regulated Trh expression. In rat fetal hypothalamic cells Klf4 regulated Trh promoter activity through CACCC and GC motifs present on the Trh gene promoter. Accordingly, hypothalamic Trh expression was down-regulated at embryonic day 15 in the Klf4−/− mice resulting in diminished bioactive peptide levels. Although at the neonatal stage the Trh transcript levels of the Klf4−/− mice were normal, the reduction in peptide levels persisted. Thus, our data indicate that Klf4 plays a key role in the maturation of TRH expression in hypothalamic neurons.

Epigenetic silencing of tumor suppressor gene promoters has become a more frequent phenomenon in cancer than previously anticipated. In this study we addressed the mechanisms involved in the protection of the p53 tumor suppressor gene against epigenetic silencing in human transformed cell lines. We characterized a binding site for the CCCTC-binding factor (CTCF) in the human p53 gene promoter that contributes to its transcriptional expression, and has the ability to maintain this regulatory element in a local open chromatin configuration. In the absence of CTCF we observe the incorporation of repressive histone marks, such as H3K9me3, H3K27me3 and H4K20me3, in different sub-domains of the upstream regulatory sequence. This evidence suggests that CTCF protects the p53 gene promoter against repressive histone marks. Notably, no apparent direct correlation between repression and DNA hypermethylation has been detected. Together, we present evidence supporting the relevant role of CTCF in the epigenetic regulation of tumor suppressor genes and cancer. We propose that CTCF is a strategic component responsible for the maintenance and segregation of epigenetic traits.

Chromatin immunoprecipitation has been widely used to determine the status of histone covalent modifications and also to investigate DNA-protein and protein-protein associations to a particular genomic location in vivo. Generally, DNA regulatory elements nucleate the interaction of several transcription factors in conjunction with ubiquitous and/or tissue-specific cofactors in order to regulate gene transcription. Therefore, it has become relevant to determine the cohabitation of several proteins in a particular developmental stage and cell type. Furthermore, multiple post-translational histone modifications can be analyzed on the same genomic location with the aim of deciphering the combinatorial pattern of histone modifications associated to specific transcriptional stages during cell commitment. Here we describe the ChIP-reChIP assay that represents a direct strategy to determine the in vivo colocalization of proteins interacting or in close contact in a chromatinized template on the basis of double and independent rounds of immunoprecipitations with high-quality ChIP grade antibodies.

The synaptonemal complex (SC) is the central key structure for meiosis in organisms undergoing sexual reproduction. During meiotic prophase I, homologous chromosomes exchange genetic information at the time they are attached to the lateral elements by specific DNA sequences. Most of these sequences, so far identified, consist of repeat DNA, which are subject to chromatin structural changes during meiotic prophase I. In this work, we addressed the effect of altering the chromatin structure of repeat DNA sequences mediating anchorage to the lateral elements of the SC. Administration of the histone deacetylase inhibitor trichostatin A into live rats caused death of cells in the pachytene stage as well as changes in histone marks along the synaptonemal complex. The most notable effect was partial loss of histone H3 lysine 27 trimethylation. Our work describes the epigenetic landscape of lateral element-associated chromatin and reveals a critical role of histone marks in synaptonemal complex integrity.

Recent reports have indicated the participation of tight junction (TJ) proteins in the regulation of gene expression and cell proliferation. Here, we have studied the role of zona occludens (ZO)-2, a TJ peripheral protein, in the regulation of cyclin D1 transcription. We found that ZO-2 down-regulates cyclin D1 transcription in a dose-dependent manner. To understand how ZO-2 represses cyclin D1 promoter activity, we used deletion analyses and found that ZO-2 negatively regulates cyclin D1 transcription via an E box and that it diminishes cell proliferation. Because ZO-2 does not associate directly with DNA, electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) assay were used to identify the transcription factors mediating the ZO-2–repressive effect. c-Myc was found to bind the E box present in the cyclin D1 promoter, and the overexpression of c-Myc augmented the inhibition generated by ZO-2 transfection. The presence of ZO-2 and c-Myc in the same complex was further demonstrated by immunoprecipitation. ChIP and reporter gene assays using histone deacetylases (HDACs) inhibitors demonstrated that HDACs are necessary for ZO-2 repression and that HDAC1 is recruited to the E box. We conclude that ZO-2 down-regulates cyclin D1 transcription by interacting with the c-Myc/E box element and by recruiting HDAC1.

Epigenetic silencing of genes relocated near telomeres, termed telomeric position effect, has been extensively studied in yeast and more recently in vertebrates. However, protection of a transgene against telomeric position effects by chromatin insulators has not yet been addressed. In this work we investigated the capacity of the chicken β-globin insulator cHS4 to shield a transgene against silencing by telomeric heterochromatin. Using telomeric repeats, we targeted transgene integration into telomeres of the chicken cell line HD3. When the chicken cHS4 insulator is incorporated to the transgene, we observe a sustained gene expression of single-copy integrants that can be maintained for >100 days of continuous culture. However, uninsulated single-copy clones showed an accelerated gene expression extinction profile. Unexpectedly, telomeric silencing was not reversed with trichostatin A or nicotidamine. In contrast, significant reactivation was obtained with 5-aza-2′-deoxycytidine, consistent with the subtelomeric DNA methylation status. Strikingly, insulated transgenes integrated into telomeric regions were enriched in histone methylation, such as H3K4me2 and H3K79me2, but not in histone acetylation. Furthermore, the cHS4 insulator counteracts telomeric position effects in an upstream stimulatory factor-independent manner. Our results suggest that this insulator has the capacity to adapt to different chromatin propagation signals in distinct insertional epigenome environments.