Journal: PLoS ONE

Article Title: Impact of the Genome on the Epigenome Is Manifested in DNA Methylation Patterns of Imprinted Regions in Monozygotic and Dizygotic Twins

doi: 10.1371/journal.pone.0025590

Figure Lengend Snippet: (A) The Position Weight Matrix (PWM) of the CTCF consensus sequence as determined by Cuddapah et al. (2009) harbours a strong preference for a cytosine residue at position 6 of the PWM. Within the region upstream the H19 gene, this cytosine is present in CTCF binding site numbers 4 and 6 (for the rs10732516 [G] allele) . The CTCF binding site number 5 within the H19 region harbours a thymidine at position 6, and is identical in sequence to CTCF binding site number 6 for the rs10732516 [A] allele (CTCF sequences are on the minus strand). (B) Analysis of CTCF binding at the H19 promoter region covering CTCF binding sites 4, 5 and 6 in six normal cell lines identified site number 5 to have minimal binding affinity within the region (ChIP-seq data from the ENCODE Chromatin Group at the Broad Institute ; UCSC Genome Browser; HMEC: human mammary epithelial cells; HSMM: normal human skeletal muscle myoblasts; HUVEC: human umbilical vein endothelial cells; NHEK: normal human epidermal keratinocytes; NHLF: normal human lung fibroblasts; H1ES: human embryonic stem cell line H1). (C) Effect of cytosine versus thymidine at position 6 of the PWM on the average enrichment score near CTCF sites within the genome of HMEC cells. Positions matching the PWM with 80% or greater sequence identity were identified and the average enrichment scores near these sites were determined −500 to +500 base pairs relative to the CTCF motif. Results for the other cell lines can be found as Figure S8. These combined results suggest a strong influence of the rs10732516 genotype on the binding affinity of CTCF at this position in the H19 -ICR.

Article Snippet: Publically available ChIP-seq data from the ENCODE Chromatin Group at the Broad Institute was downloaded for all available normal cell lines (HMEC [human mammary epithelial cells], HSMM [normal human skeletal muscle myoblasts], HUVEC [human umbilical vein endothelial cells], NHEK [normal human epidermal keratinocytes], NHLF [normal human lung fibroblasts] and the H1 human embryonic stem cell line).

Techniques: Sequencing, Residue, Binding Assay, ChIP-sequencing

Journal: Human Genetics and Genomics Advances

Article Title: Using a modular massively parallel reporter assay to discover context-dependent regulatory activity in type 2 diabetes-linked noncoding regions

doi: 10.1016/j.xhgg.2026.100606

Figure Lengend Snippet: HNF1 transcription factor motifs contribute to enhancer activity near selected T2D-associated variants (A) For 47 HNF1-motif-overlapping fragments with significant INS promoter-bias effects on activity, we designed three versions: original (motif intact), deleted (motif removed and sequence adjusted), and shuffled (dinucleotide-shuffled motif). When the tested variant was adjacent to the motif, we synthesized both reference and alternative alleles for each version. For variants directly overlapping the motif, we generated only one deletion and one shuffled fragment. (B) We synthesized four fragments corresponding to the variant rs1635852, which overlaps an HNF1 motif at a high-information-content position. The T2D risk allele (T) disrupts this motif, while the non-risk allele (C) matches the consensus. (C) Shuffling the motif significantly decreased enhancer activity compared to intact fragments with either the risk T (Wilcoxon rank-sum test p = 0.016) or non-risk C ( p = 0.008) allele. Motif deletion also significantly decreased enhancer activity compared to the non-risk C allele ( p = 0.016). (D) For the variant rs11819995, located 11 bp upstream of an HNF1 motif, we synthesized six fragments. (E) Deletion of the motif significantly decreased enhancer activity for both the reference (C, non-risk) and alternative (T, risk) alleles ( p = 0.008 for both alleles). Shuffling the motif likewise reduced activity for both alleles ( p = 0.008 for the reference allele and p = 0.056 for the alternative allele). (F) To assess context-specific effects, we cloned these fragments into MPRA vectors with the SCP1 or skeletal-muscle-specific MYBPC2 promoter and delivered all three to LHCN-M2 human skeletal muscle myotubes ( n = 6). (G) When paired with the INS promoter, the shuffled rs11819995-containing fragment showed increased activity relative to the original fragment ( p = 0.015); however, none of the fragments containing rs11819995 functioned as enhancers in LHCN-M2 myotubes, regardless of promoter context. Overall, their activity is highest when paired with the skeletal-muscle-specific promoter.

Article Snippet: We obtained INS-1 832/13 rat insulinoma cells from Dr. Christopher Newgard (Sarah W. Stedman Nutrition and Metabolism Center, Duke University, Durham, NC) and LHCN-M2 human skeletal muscle myoblasts from Evercyte.

Techniques: Activity Assay, Sequencing, Variant Assay, Synthesized, Generated, Clone Assay