A Distal CCAAT/NUCLEAR FACTOR Y Complex Promotes Chromatin Looping at the FLOWERING LOCUS T Promoter and Regulates the Timing of Flowering in Arabidopsis

For many plant species, reproductive success relies on the proper timing of flowering, and photoperiod provides a key environmental input. Photoperiod-dependent flowering depends on timely expression of FLOWERING LOCUS T (FT); however, the coordination of various cis-regulatory elements in the FT promoter is not well understood. Here, we provide evidence that long-distance chromatin loops bring distal enhancer elements into close association with the proximal promoter elements bound by CONSTANS (CO). Additionally, we show that NUCLEAR FACTOR Y (NF-Y) binds a CCAAT box in the distal enhancer element and that CCAAT disruption dramatically reduces FT promoter activity. Thus, we propose the recruitment model of photoperiod-dependent flowering where NF-Y complexes, bound at the FT distal enhancer element, help recruit CO to proximal cis-regulatory elements and initiate the transition to reproductive growth.     

Polycomb Domain Formation Depends on Short and Long Distance Regulatory Cues

Background

Polycomb group (PcG) proteins dynamically define cellular identities through the epigenetic repression of key developmental genes. In Drosophila, cis-regulatory regions termed PcG response elements (PREs) act as nucleation sites for PcG proteins to create large repressive PcG domains that are marked by trimethylation of lysine 27 on histone H3 (H3K27me3). In addition to an action in cis, PREs can interact over long distances, thereby enhancing PcG dependent silencing. How PcG domains are established, which factors limit their propagation in cis, and how long range interactions of PREs in trans affect the chromatin structure is largely unknown.

Principal Findings

We demonstrate that the insertion of a PRE-containing transgene in the Drosophila genome generates an artificial PcG domain and we analyze its organization by quantitative ChIP and ChIP-on-chip experiments. Intriguingly, a boundary element and known insulator proteins do not necessarily interfere with spreading of H3K27me3. Instead, domain borders correlate with the presence of promoter regions bound by RNA Polymerase II and active chromatin marks. In contrast, genes that are silent during early fly development get included within the PcG domain and this incorporation interferes with gene activation at later developmental stages. Moreover, trans-interaction of the transgenic PRE with its homologous endogenous PRE results in increased PcG binding, correlating with reinforced silencing of genes within the domain borders.

Conclusions

Our results suggest that higher-order organization of PcG-bound chromatin can stabilize gene silencing within PcG domains. Further we propose that multi-protein complexes associated with active promoters are able to define the limits of PcG domains. Future work aimed to pinpoint the factors providing this barrier function will be required to understand the precise molecular mechanism by which active promoter regions can act as boundaries to stop spreading of H3K27me3.     

Sequence characterization and promoter identification of porcine APC10 gene

APC10 protein, a subunit of the anaphase-promoting complex (APC), plays an essential role in the progression of cells from mitosis to G1. In this study, we cloned and sequenced partial cDNA, intron 1 and 5′-flanking sequences of porcine APC10. The partial cDNA is 595 bp long and has an open reading frame of 558 bp which encodes 185 putative animo acids. Real-time PCR analysis revealed that the porcine APC10 mRNA expression shows a wide distribution and expression levels varies within a small different range in detected tissues. The deduced protein has a high identity with other eight species and comprises a conserved DOC domain. The phylogenetic tree indicated that porcine APC10 has the closest genetic relationship with human, monkey and dog. Promoter activity was demonstrated by transient transfection of 5′-deletion promoter/luciferase constructs into PK15 cells, and indicated that the proximal region from ?2,052 to ?1,764 is necessary for basal promoter activity. Positive cis-regulatory elements are present from ?2,544 to ?2,052 and from ?3,114 to ?2,774, while negative cis-regulatory elements may be present from ?2,774 to ?2,544. Yeast one-hybrid assay revealed Sp1 can interact with proximal promoter region of porcine APC10.