AUTHOR=Cortés-Fernández de Lara Josué , Núñez-Martínez Hober Nelson , Tapia-Urzúa Gustavo , Garza-Manero Sylvia , Peralta-Alvarez Carlos Alberto , Furlan-Magaril Mayra , González-Buendía Edgar , Escamilla-Del-Arenal Martín , Casasola Andrea , Guerrero Georgina , Recillas-Targa Felix TITLE=A novel cis-regulatory element regulates αD and αA-globin gene expression in chicken erythroid cells JOURNAL=Frontiers in Genetics VOLUME=15 YEAR=2024 URL=https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2024.1384167 DOI=10.3389/fgene.2024.1384167 ISSN=1664-8021 ABSTRACT=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.