Studying physical chromatin interactions in plants using Chromosome Conformation Capture (3C)

Louwers, Marieke; Splinter, Erik; Driel, Roel van; Laat, Wouter de; Stam, Maike

doi:10.1038/nprot.2009.113

Cited by 90 publications

(99 citation statements)

References 24 publications

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“…Two clear peaks were consistently measured, corresponding to interactions with DpnII-released fragments IV and VII ( Figure 3B; note that fragment VII corresponds to the 25.3-kb CCAAT site). We also inconsistently measured an interaction with fragment III, but this is likely due to linkage and incomplete digestion as previously described (Louwers et al, 2009a(Louwers et al, , 2009b. To address whether or not these putative DNA loops depended on CO or an NF-Y complex, we repeated the 3C experiment using the low FTexpressing, late-flowering, strong hypomorphic mutant co-9 (also referred to as co-sail; Laubinger et al, 2006) and nfyb2 nf-yb3.…”

Section: Co-and Nf-y-dependent Chromatin Loops Form In a Time-dependementioning

confidence: 99%

“…Using the chromatin conformational capture (3C) technique (Hagège et al, 2007;Louwers et al, 2009aLouwers et al, , 2009b, we addressed whether DNA loops bring the CORE1/2 and 25.3-kb CCAAT sites within close proximity. With CORE1/2 acting as the anchor point, we sequentially queried cross-linking interactions between this region and various DpnII restriction enzyme-released, CCAATcontaining DNA fragments in the functionally defined ;6.5-kb FT promoter ( Figure 3A).…”

Section: Co-and Nf-y-dependent Chromatin Loops Form In a Time-dependementioning

confidence: 99%

“…3C experiments were performed according to the method described for maize (Zea mays) with minor alterations (Louwers et al, 2009a). In short, leaves from 2-week-old LD-grown Arabidopsis were cross-linked using formaldehyde and nuclei were isolated.…”

Section: C-qpcrmentioning

confidence: 99%

“…To this end, the ligation product of an unrelated locus was used in the normalization of the qPCR data. EF-1a was identified to fulfill the required criteria (Czechowski et al, 2005;Louwers et al, 2009a). To control for differences in primer set efficiency during PCR amplification, a control template was required that contains all possible ligation products of the loci of interest (FT and EF1a) in equimolar amounts.…”

Section: C-qpcrmentioning

confidence: 99%

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

et al. 2014

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ORCID ID: 0000-0001-7182-9594 (B.F.H.)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.

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Section: Co-and Nf-y-dependent Chromatin Loops Form In a Time-dependementioning

confidence: 99%

Section: Co-and Nf-y-dependent Chromatin Loops Form In a Time-dependementioning

confidence: 99%

Section: C-qpcrmentioning

confidence: 99%

Section: C-qpcrmentioning

confidence: 99%

See 2 more Smart Citations

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

et al. 2014

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“…incubated at room temperature for 5 min under vacuum in MC buffer with 0.15 M glycine. Approximately 2 g fixed tissue was homogenized with liquid nitrogen and resuspended in nuclei isolation buffer (Louwers et al 2009) and filtered with a 40-nm cell strainer. The procedures for enriching nuclei from flow-through and subsequent denaturation were done according to a 3C protocol established for maize (Louwers et al 2009).…”

Section: Hi-c Library Preparationmentioning

confidence: 99%

Genome-wide analysis of local chromatin packing in Arabidopsis thaliana

et al. 2014

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The spatial arrangement of interphase chromosomes in the nucleus is important for gene expression and genome function in animals and in plants. The recently developed Hi-C technology is an efficacious method to investigate genome packing. Here we present a detailed Hi-C map of the three-dimensional genome organization of the plant Arabidopsis thaliana. We find that local chromatin packing differs from the patterns seen in animals, with kilobasepair-sized segments that have much higher intrachromosome interaction rates than neighboring regions, representing a dominant local structural feature of genome conformation in A. thaliana. These regions, which appear as positive strips on two-dimensional representations of chromatin interaction, are enriched in epigenetic marks H3K27me3, H3.1, and H3.3. We also identify more than 400 insulator-like regions. Furthermore, although topologically associating domains (TADs), which are prominent in animals, are not an obvious feature of A. thaliana genome packing, we found more than 1000 regions that have properties of TAD boundaries, and a similar number of regions analogous to the interior of TADs. The insulator-like, TAD-boundary-like, and TAD-interior-like regions are each enriched for distinct epigenetic marks and are each correlated with different gene expression levels. We conclude that epigenetic modifications, gene density, and transcriptional activity combine to shape the local packing of the A. thaliana nuclear genome.

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Wild‐type p53 regulates OTOP2 transcription through DNA loop alteration of the promoter in colorectal cancer

et al. 2018

FEBS Open Bio

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Colorectal cancer ( CRC ) is the third most commonly diagnosed malignancy worldwide and remains a major public health issue. Therefore, further investigation is required to delineate the cellular and molecular mechanisms underlying colorectal tumorigenesis. Using CRC data taken from The Cancer Genome Atlas, we determined that the expression of otopetrin 2 ( OTOP 2) is highly correlated with malignancy grade and rate of patient survival. Here, we report that OTOP 2 is down‐regulated in cancerous tissues and that elevated OTOP 2 effectively suppresses tumor proliferation in vitro . We demonstrate that wild‐type p53 (wtp53), but not mutant p53 (mtp53), can regulate the transcription of otop2 in CRC cells. Subsequently, we investigate the chromatin architecture of the otop2 promoter, whereby we discover alterations in p53‐dependent DNA loop organization and CCCTC ‐binding factor ( CTCF ) binding between cells with wtp53 and mtp53. In conclusion, our study promotes an in‐depth understanding of tumorigenesis, which may also lead to the development of therapeutic applications targeting human malignancy.

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Studying physical chromatin interactions in plants using Chromosome Conformation Capture (3C)

Cited by 90 publications

References 24 publications

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

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

Genome-wide analysis of local chromatin packing in Arabidopsis thaliana

Wild‐type p53 regulates OTOP2 transcription through DNA loop alteration of the promoter in colorectal cancer

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