A novel cis-regulatory element regulates αD and αA-globin gene expression in chicken erythroid cells.

Background: Cis-regulatory elements (CREs) play crucial roles in regulating gene expression during erythroid cell differentiation. Genome-wide erythroid-specific CREs have not been characterized in chicken erythroid cells, which is an organism model used to study epigenetic regulation during erythropoiesis. Methods: Analysis of public genome-wide accessibility (ATAC-seq) maps, along with transcription factor (TF) motif analysis, CTCF, and RNA Pol II occupancy, as well as transcriptome analysis in fibroblasts and erythroid HD3 cells, were used to characterize erythroid-specific CREs. An α-globin CRE was identified, and its regulatory activity was validated in vitro and in vivo by luciferase activity and genome-editing assays in HD3 cells, respectively. Additionally, circular chromosome conformation capture (UMI-4C) assays were used to distinguish its role in structuring the α-globin domain in erythroid chicken cells. Results: Erythroid-specific CREs displayed occupancy by erythroid TF binding motifs, CTCF, and RNA Pol II, as well as an association with genes involved in hematopoiesis and cell differentiation. An α-globin CRE, referred to as CRE-2, was identified as exhibiting enhancer activity over αD and αA genes in vitro and in vivo. Induction of terminal erythroid differentiation showed that α-globin CRE-2 is required for the induction of αD and αA. Analysis of TF binding motifs at α-globin CRE-2 shows apparent regulation mediated by GATA-1, YY1, and CTCF binding. Conclusion: Our findings demonstrate that cell-specific CREs constitute a key mechanism that contributes to the fine-tuning gene regulation of erythroid cell differentiation and provide insights into the annotation and characterization of CREs in chicken cells.

Chromatin remodeling effects on enhancer activity.

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.

A novel chromatin insulator regulates the chicken folate receptor gene from the influence of nearby constitutive heterochromatin and the ß-globin locus.

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 ß-globin gene cluster, which are separated by 16kb 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 ß-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 16kb of constitutive heterochromatin contributes to silencing of the FOLR1 gene during erythroid differentiation.

The Gpr1/Zdbf2 locus provides new paradigms for transient and dynamic genomic imprinting in mammals.

Many loci maintain parent-of-origin DNA methylation only briefly after fertilization during mammalian development: Whether this form of transient genomic imprinting can impact the early embryonic transcriptome or even have life-long consequences on genome regulation and possibly phenotypes is currently unknown. Here, we report a maternal germline differentially methylated region (DMR) at the mouse Gpr1/Zdbf2 (DBF-type zinc finger-containing protein 2) locus, which controls the paternal-specific expression of long isoforms of Zdbf2 (Liz) in the early embryo. This DMR loses parental specificity by gain of DNA methylation at implantation in the embryo but is maintained in extraembryonic tissues. As a consequence of this transient, tissue-specific maternal imprinting, Liz expression is restricted to the pluripotent embryo, extraembryonic tissues, and pluripotent male germ cells. We found that Liz potentially functions as both Zdbf2-coding RNA and cis-regulatory RNA. Importantly, Liz-mediated events allow a switch from maternal to paternal imprinted DNA methylation and from Liz to canonical Zdbf2 promoter use during embryonic differentiation, which are stably maintained through somatic life and conserved in humans. The Gpr1/Zdbf2 locus lacks classical imprinting histone modifications, but analysis of mutant embryonic stem cells reveals fine-tuned regulation of Zdbf2 dosage through DNA and H3K27 methylation interplay. Together, our work underlines the developmental and evolutionary need to ensure proper Liz/Zdbf2 dosage as a driving force for dynamic genomic imprinting at the Gpr1/Zdbf2 locus.

Jarid2 Is Implicated in the Initial Xist-Induced Targeting of PRC2 to the Inactive X Chromosome.

During X chromosome inactivation (XCI), the Polycomb Repressive Complex 2 (PRC2) is thought to participate in the early maintenance of the inactive state. Although Xist RNA is essential for the recruitment of PRC2 to the X chromosome, the precise mechanism remains unclear. Here, we demonstrate that the PRC2 cofactor Jarid2 is an important mediator of Xist-induced PRC2 targeting. The region containing the conserved B and F repeats of Xist is critical for Jarid2 recruitment via its unique N-terminal domain. Xist-induced Jarid2 recruitment occurs chromosome-wide independently of a functional PRC2 complex, unlike at other parts of the genome, such as CG-rich regions, where Jarid2 and PRC2 binding are interdependent. Conversely, we show that Jarid2 loss prevents efficient PRC2 and H3K27me3 enrichment to Xist-coated chromatin. Jarid2 thus represents an important intermediate between PRC2 and Xist RNA for the initial targeting of the PRC2 complex to the X chromosome during onset of XCI.

Evolutionary diversity and developmental regulation of X-chromosome inactivation.

X-chromosome inactivation (XCI) results in the transcriptional silencing of one X-chromosome in females to attain gene dosage parity between XX female and XY male mammals. Mammals appear to have developed rather diverse strategies to initiate XCI in early development. In placental mammals XCI depends on the regulatory noncoding RNA X-inactive specific transcript (Xist), which is absent in marsupials and monotremes. Surprisingly, even placental mammals show differences in the initiation of XCI in terms of Xist regulation and the timing to acquire dosage compensation. Despite this, all placental mammals achieve chromosome-wide gene silencing at some point in development, and this is maintained by epigenetic marks such as chromatin modifications and DNA methylation. In this review, we will summarise recent findings concerning the events that occur downstream of Xist RNA coating of the inactive X-chromosome (Xi) to ensure its heterochromatinization and the maintenance of the inactive state in the mouse and highlight similarities and differences between mammals.

Promoter characterization and role of CRE in the basal transcription of the rat SNAT2 gene.

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.

GATA-1 modulates the chromatin structure and activity of the chicken alpha-globin 3' enhancer.

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.

Globin genes transcriptional switching, chromatin structure and linked lessons to epigenetics in cancer: a comparative overview.

At the present time research situates differential regulation of gene expression in an increasingly complex scenario based on interplay between genetic and epigenetic information networks, which need to be highly coordinated. Here we describe in a comparative way relevant concepts and models derived from studies on the chicken alpha- and beta-globin group of genes. We discuss models for globin switching and mechanisms for coordinated transcriptional activation. A comparative overview of globin genes chromatin structure, based on their genomic domain organization and epigenetic components is presented. We argue that the results of those studies and their integrative interpretation may contribute to our understanding of epigenetic abnormalities, from beta-thalassemias to human cancer. Finally we discuss the interdependency of genetic-epigenetic components and the need of their mutual consideration in order to visualize the regulation of gene expression in a more natural context and consequently better understand cell differentiation, development and cancer.

YY1 and GATA-1 interaction modulate the chicken 3'-side alpha-globin enhancer activity.

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.