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.

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.

Transcriptome-wide identification of in vivo interactions between RNAs and RNA-binding proteins by RIP and PAR-CLIP assays.

Comprehensive genomic and computational studies in the era of high-throughput sequencing revealed that the major proportion of the human genome is transcribed. This novel insight confronted the scientific community with new questions concerning the expanded role of RNA, especially noncoding RNA (ncRNA), in cellular pathways. In recent years, there has been mounting evidence that ncRNAs and RNA binding proteins (RBPs) are involved in a wide range of biological processes, such as developmental transitions, cell differentiation, stress response, genome organization, and regulation of gene expression. In particular, in the chromatin field long noncoding RNAs (lncRNAs) have drawn increasing attention to their function in epigenetic regulation due to the fact that they were found to interact with multiple chromatin regulators and modifiers. Recently, techniques to study the extent of RNA-protein interactions have been developed in many research laboratories. Here we describe protocols for RNA Immunoprecipitation-Sequencing (RIP-Seq) and Photoactivatable-Ribonucleoside-Enhanced Cross-linking and Immunoprecipitation combined with deep sequencing (PAR-CLIP-Seq) to identify RNA targets of RNA-binding proteins (RBPs) on a transcriptome-wide level, discussing advantages and drawbacks.