Spatial organization of chromatin of KLF5 gene promoter region in pancreatic ductal adenocarcinoma cells

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Abstract

Pancreatic Ductal AdenoCarcinoma (PDAC) is characterized by a poor prognosis and is poorly amenable to modern therapies. A range of cell cultures reflecting different degrees of tumor differentiation and malignancy can serve as a model of PDAC development. Highly differentiated low malignant cells are characterized by increased expression of the KLF5 gene. The KLF5 protein is a vivid representative of multifunctional transcription factors, and its involvement in a variety of cellular processes, particularly in the pathology of various cancers, has been demonstrated. We investigated the spatial organization of chromatin of regulatory regions of KLF5 gene using highly differentiated Capan2 cells PDAC with a high level of KLF5 expression and poorly differentiated MIA PaCa2 PDAC cells with a low level of this gene expression by circular chromosome conformation capture (4C-seq). It was shown that the number and distribution of contacts of the KLF5 regulatory region with other chromatin regions are significantly different for these cell types; the number of contacts is significantly higher for Capan2 cells. There is a correlation between the expression level of genes close to KLF5 and the intensity of their sequence contacts with the KLF5 regulatory region, indicating that their expression is coordinated, possibly within the transcriptional factory. Only Capan2 is characterized by a high level of contacts of the KLF5 regulatory region with the gene free region containing a cluster of PDAC-associated single nucleotide polymorphisms (SNP). Thus, the total number of contacts of the promoter region of the KLF5 gene and the expression level of most of the surrounding KLF5 genes decrease as the grade of cell malignancy increases.

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M. V. Zinovyeva

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: lev@ibch.ru
Russian Federation, Moscow, 117997

L. G. Nikolaev

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Author for correspondence.
Email: lev@ibch.ru
Russian Federation, Moscow, 117997

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10. Fig. 1. Structure and sequencing of libraries. a – Structure of libraries. P5, P7, Reading – sequences for Illumina sequencing; H- and D-primers – unique primers for inverse PCR from the side of the Hind III and Dpn II sites, respectively; index – index sequences; insert – amplified genomic sequence in contact with the anchor. б – Electrophoresis in 4C agarose gel of libraries after the second round of PCR amplification. The library number corresponds to the Hind III primer number (see Table 1). M_1 and M_0.1 – DNA markers of 1 and 0.1 kb, respectively. в – Typical arrangement of reads relative to the Hind III cleavage sites in the region of chromosome 13. Three ligation options are shown: (1) along one of the strands; (2) along the opposite strand; (3) on both strands with different efficiencies. Figure prepared using SeqMonk (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk). г – Location of contact sites in a 6-Mb region of chromosome 13 around the KLF5 gene. The ordinate axis shows the number of reads per million (RPM) in the corresponding library, the blue vertical line shows the position of the anchor sequences. The top two lanes show the distribution of contacts of the K1 anchor sequence and Capan2 cells in two independent experiments; the next two lanes show the same data for MIA PaCa2 cells. д – Location of contact sites in a small (200 kb) region of chromosome 13 near the KLF12 gene. The ordinate axis shows the number of reads in the corresponding library. The top two lanes compare the distribution of contacts of the K1 and K3 anchor sequences in Capan2 cells, the next two lanes do the same for MIA PaCa2 cells. The bottom shows the location of Hind III cleavage sites.

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11. Fig. 2. Distribution of contacts of the KLF5 gene anchor region in a 6-Mb region of human chromosome 13 (3 Mb on the 5´- and 3´-sides from the anchor). The results of the analysis of MIA PaCa2 and Capan2 cells obtained using peakC, FourCSeq, and R3Cseq are shown and presented as –log(P-value). The horizontal line corresponds to the threshold P-value (0.05 for peakC and 0.01 for FourCseq and R3Cseq). For differential contacts detected using the 4C-ker program, the threshold P-value was 0.01. The vertical arrow shows the position of the anchor sequence. The bottom panel shows the location of eight topologically associated domains (TADs) of chromatin according to Hi-C data for Capan1 cells [44].

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12. Fig. 3. The arrangement of selected and normalized contact sites of the KLF5 gene anchor region in the 6-Mb region of human chromosome 13 relative to some functional elements. The contact regions specific to Capan2 and MIA PaCa2 cells and common to both cell lines are shown separately. a – Tissue specificity of gene expression in Capan2 and MIA PaCa2 cells, the relationship between the gene expression level and the number of its contacts with the KLF5 promoter region. The ordinate axes show the FPKM values ​​for assessing the gene expression level and –lg(P-value) for the contact sites. б – The arrangement relative to the contacts of single nucleotide polymorphism points and deletions/insertions (SNPs/indels) associated with PDAC according to the genome-wide analysis (GWAS, Genome-Wide Association Studies, see [11, 13]).

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13. Fig. 4. Relationship between gene expression levels and intensity of their contacts with the KLF5 anchor region in two gene-rich regions of chromosome 13 (circled by squares in Fig. 3a). a – Region with coordinates 75,800,000 – 76,500,000 bp; б – Region with coordinates 73,280,000 – 73,600,000 bp. The ordinate axes show FPKM values ​​for gene expression levels and –log(P-value) for contact regions. Arrows indicate the direction of gene transcription. Gene expression levels are given according to data from [7] (available in the NCBI GEO database, GSE64558). All contacts identified in each cell line (both cell line-specific and common) are shown.

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14. Fig. 5. Distribution of genomic contacts of the KLF5 gene anchor region and histone H3 modifications in the chromatin of Capan2 and MIA PaCa2 cells (available in the NCBI GEO database, GSE64560) in a 6-Mb region of chromosome 13. The ordinate axes show FPKM values ​​for gene expression levels and –log(P-value) for contact sites. Gene expression levels are given according to [7] (available in the NCBI GEO database, GSE64560). All contacts identified in each cell line (both line-specific and common) are shown.

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