Summary• CONSTANS is an evolutionarily-conserved central component of the genetic pathway that controls the onset of flowering in response to daylength. However, the specific biochemical mechanism by which the CONSTANS protein regulates the expression of its target genes remains largely unknown.• By using a combination of cell-based expression analysis and in vitro DNA binding studies, we have demonstrated that CONSTANS possesses transcriptional activation potential and is capable of directly binding to DNA.• CONSTANS was found to bind DNA via a unique sequence element containing a consensus TGTG(N2-3)ATG motif. This element is present in tandem within the FLOWERING LOCUS T promoter and is sufficient for CO binding and activity. The conserved CCT (CONSTANS, CONSTANS-like and TOC1) domain of CONSTANS was shown to be required for its recruitment to the DNA motif and other CCTcontaining proteins were also found to have the ability to regulate gene expression via this element.• The CCAAT box, which has been previously hypothesized as a recruitment site for complexes containing the CONSTANS protein, potentiated CONSTANSmediated activation but was not essential for CONSTANS recruitment to a target promoter or for its activity as a transcriptional factor.

a b s t r a c t a r t i c l e i n f oIron is essential for the human being, involving in oxygen transport, energy metabolism and DNA synthesis. Iron homeostasis is tightly governed by the hepcidin-ferroportin axis, of which hepcidin is the master regulator. Excess iron is associated with various diseases including osteopenia and osteoporosis, which are closely related to the alternation of the endogenous estrogen level. To verify the biological effect of estrogen on iron metabolism, we established a mouse model of estrogen deficiency by ovariectomy. We demonstrated that the hemoglobin content and serum iron level decreased, whereas the tissue iron level in liver and spleen increased in the ovariectomized mice. Moreover, the transcription of hepatic hepcidin was elevated in ovariectomized mice compared to the control mice. We further demonstrated that there was an estrogen response element (ERE) in the promoter region of the hepcidin gene. The assay using the luciferase reporter system confirmed the existence of a functional ERE in the hepcidin promoter, as the estradiol treatment reduced hepcidin expression in cells transfected with ERE-intact construct, with no response to estradiol in cells transfected with ERE-devoid construct. In conclusion, estrogen greatly contributes to iron homeostasis by regulating hepatic hepcidin expression directly through a functional ERE in the promoter region of hepcidin gene. These findings might help build a better understanding towards the etiology of postmenopausal osteoporosis accompanied by excess tissue iron (such as iron retention of osteoclasts in bone) under estrogen deficiency.

Purpose Current clinical genomics assays primarily utilize short-read sequencing (SRS), but SRS has limited ability to evaluate repetitive regions and structural variants. Long-read sequencing (LRS) has complementary strengths, and we aimed to determine if LRS could offer a means to identify overlooked genetic variation in patients undiagnosed by SRS. Methods We performed low coverage genome LRS to identify structural variants in a patient who presented with multiple neoplasia and cardiac myxomata, in whom targeted clinical testing and genome SRS were negative. Results This LRS approach yielded 6,971 deletions and 6,821 insertions >50bp. Filtering for variants that are absent in an unrelated control and overlap a disease gene coding exon identified three deletions and three insertions. One of these, a heterozygous 2,184 bp deletion, overlaps the first coding exon of PRKAR1A, which is implicated in autosomal dominant Carney complex. RNA sequencing demonstrated decreased PRKAR1A expression. The deletion was classified as pathogenic based on guidelines for interpretation of sequence variants. Conclusions This first successful application of genome LRS to identify a pathogenic variant in a patient suggests that LRS has significant potential to identify disease-causing structural variation. Larger studies will be ultimately required to evaluate the potential clinical utility of LRS.