The amount of DNA amplified from immunoprecipitated DNA was normalized to that amplified from input DNA. Chromatin was immunoprecipitated with anti-DYK (FLAG) antibody or nonspecific antibody. KRAS was used to identify non-specific interactions. ( A) Quantitative ChIP-PCR analysis of PAX5 occupancy of the GRHL1 and GRH元 regulatory regions was performed in HEK293 cells transfected with pcDNA3.1-N-DYK-PAX5. Where indicated, 3 μg anti-KLF4 antibody was added per lane (ab106629, Abcam). Cold probe: unlabeled double-stranded oligonucleotides including -409/-400 (left panel, lane 3) or -409/-400 with SNP rs115898376 (C/T) (left panel, lane 6) or -650/-641 (central panel, lane 3) or -1302/-1293 (right panel, lane 3) regions of GRHL genes (100-fold molar excess of competitors). ( B) EMSA analysis performed with probes including KLF4 binding sequences: left panel–in the region -409/-400 (lane 2) or -409/-400 with the minor frequency allele of SNP rs115898376 (C/T) (lane 5) of the GRHL1 promoter central panel–in the region -650/-641 (lane 2) of the GRHL2 promoter right panel–in the region -1302/-1293 (lane 2) of the GRH元 promoter. Data are shown as means ± SEM from experiments independently performed in triplicate, *significantly different at p≤ 0.05. Chromatin was immunoprecipitated with anti-KLF4 antibody or nonspecific antibody. ZNF333 was used to identify non-specific interactions. ( A) Quantitative ChIP-PCR analysis of KLF4 occupancy of GRHL1, GRHL2 and GRH元 regulatory regions was performed in HEK293 cells transfected with pcDNA3.1-HA-KLF4 FL. ( D) Multi-alignments around the most conserved instances of motifs KLF4 and PAX5. The most conserved motif instances are marked by orange rectangles. ( C) Evolutionarily conserved TFBS motif instances for KLF4 and PAX5 identified using MotEvo with the default parameters. In this file, all the analysis parameters are provided as example values, viewable and usable upon opening the client in Taverna Workbench. The plots were generated using the webservice of the NGD database, accessed with the client PlotGenomic.t2flow, which is provided as S1 File. Instances of SwissRegulon TFBS motifs for KLF4 and PAX5 in the promoter taken from the NGD database are also shown. ( B) Analysis windows used for multi-alignment, based on promoter features (DNase I-seq, CpG, H3K9ac, H3K4me3) from Ensembl v.79 are marked as blue rectangles within the ± 10 kb flanks of the TSS. ( A) The overall analysis workflow, starting from the selection of the two transcription factors of interest that was based on the literature (PubMed). The evidence presented here suggests an important role of KLF4 and PAX5 in the regulation of expression of GRHL1-3 genes. The presence of minor frequency allele of single nucleotide polymorphism rs115898376 in the promoter of the GRHL1 gene affected the binding of KLF4 to this site. The levels of GRHL1 and GRH元 expression were decreased by 30% or 33% in PAX5-overexpressing HEK293 cells. In KLF4-overexpressing HEK293 cells, the expression of GRHL1 and GRH元 genes was upregulated by 32% and 60%, respectively, whereas the mRNA level of GRHL2 gene was lowered by 28% when compared to the respective controls. Ectopic expression of KLF4 or PAX5 alters the expression of GRHL genes. Here we report that the Krüppel-like factor 4 (KLF4) and the paired box 5 factor (PAX5) bind to the regulatory regions of the GRHL genes and regulate their expression. ![]() Previously, there were no systematic analyses of the promoters of GRHL genes or transcription factors that bind to these promoters. However, little is known about the regulation of expression of GRHL genes. These genes are also very important in the development of many types of cancer. Genes from the Grainyhead-like (GRHL) family code for transcription factors necessary for the development and maintenance of various epithelia.
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