Table 3 A summary of findings on the analysis of promoter regulatory regions of the chicken Ovalbumin gene

Strategy

In vitro gene transfer of OvCAT fusion genes containing mutations in the SDRE and NRE

In vitro gene transfer of OvCAT fusion genes containing truncated Ovalbumin promoter with various lengths of the NRE and mutation in the silencer

In vitro gene transfer of OvCAT fusion genes containing mutations in the SDRE

In vivo gene transfer of OvCAT fusion genes containing various lengths of truncated Ovalbumin promoter

In vitro gene transfer of OvCAT fusion genes containing mutations in the SDRE

In vitro gene transfer of OvCAT fusion genes containing truncated Ovalbumin with deletions in the NRE, the SDRE, and mutations in the COUP, and overexpression of COUP-TF1

In vitro gene transfer of OvCAT fusion genes containing truncated Ovalbumin promoter with deletions in the NRE, the SDRE, and mutations in the CAR and the silencer

In vitro gene transfer of CRISPR excision for In situ deletion of the genomic SDRE/NRE and CRISPR HDR for insertion of promoter-less reporter

Cell type

Primary oviduct cells

Primary oviduct cells

Primary oviduct cells

Oviduct and liver of laying hens

Primary oviduct cells

Primary oviduct cells

LMH/2A cell line

DF1 cell line

Findings

Induction of the Ovalbumin gene by steroid hormones requires complex interactions involving both the SDRE and NRE

The NRE is a multifunctional regulatory element containing at least two sites for induction by steroids and three elements that repress Ovalbumin transcription

The Ovalbumin gene is regulated by steroid Hormones, binding to a DNA element from -891 to -878 in the SDRE

The Ovalbumin gene promoter region between -3200 and -2800 bp (a tissue-specific silencer-like) represses the Ovalbumin gene transcription in the liver, but not in the oviduct of laying hens

The NRE contains not only the sites responsible for the repression of the gene but also a positive element that is required for the responsiveness to steroid hormones

Without the NRE, the SDRE is sufficient for induction by estrogen, irrespective of the COUP site. with the NRE intact, the COUP site is required for steroid induction. Without the NRE, the COUP site attenuates transcriptional activity

The inhibition of Ovalbumin gene expression in non-oviduct cells is a combination of the lack of essential positive factors and the presence of an active repressor,which binds to the CAR element

In situ genomic deletion of the SDRE and NRE is sufficient to derepress the transcription of the Ovalbumin gene and induced the activity of an inserted transgene in the non-oviduct cells

Comment

Although previous studies have provided insights into the mechanisms that underlie the hormonal, and tissue-specific regulation of Ovalbumin gene expression, most have applied plasmid-based methods, irrespective of the genome context. Combining genomics or transcriptomics approaches with plasmid-based MPRA (massively parallel reporter assays) and CRISPR-based in vivo methods can develop our understanding of the mechanisms underlying regulatory events of gene expression. In this study, to consider the genomic context, we have applied CRISPR tools to manipulate the genomic regulatory regions of the Ovalbumin promoter