Fig 1.
Bioinformatics predictions of TFBS motifs for KLF4 and PAX5 in the promoters of human GRHL1-3 genes.
(A) The overall analysis workflow, starting from the selection of the two transcription factors of interest that was based on the literature (PubMed). (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. Instances of SwissRegulon TFBS motifs for KLF4 and PAX5 in the promoter taken from the NGD database [27] are also shown. The plots were generated using the webservice of the NGD database [27], accessed with the client PlotGenomic.t2flow, which is provided as S1 File. In this file, all the analysis parameters are provided as example values, viewable and usable upon opening the client in Taverna Workbench [28] http://www.taverna.org.uk/download/workbench/. (C) Evolutionarily conserved TFBS motif instances for KLF4 and PAX5 identified using MotEvo [30] with the default parameters. The most conserved motif instances are marked by orange rectangles. (D) Multi-alignments around the most conserved instances of motifs KLF4 and PAX5.
Fig 2.
KLF4 binds to the regulatory regions of GRHL genes.
(A) Quantitative ChIP-PCR analysis of KLF4 occupancy of GRHL1, GRHL2 and GRHL3 regulatory regions was performed in HEK293 cells transfected with pcDNA3.1-HA-KLF4 FL. ZNF333 was used to identify non-specific interactions. Chromatin was immunoprecipitated with anti-KLF4 antibody or nonspecific antibody. The amount of DNA amplified from immunoprecipitated DNA was normalized to that amplified from input DNA. Data are shown as means ± SEM from experiments independently performed in triplicate, *significantly different at p≤ 0.05. (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 GRHL3 promoter. Lane 1: probe only. 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). Where indicated, 3 μg anti-KLF4 antibody was added per lane (ab106629, Abcam).
Fig 3.
PAX5 binds to the regulatory regions of GRHL genes.
(A) Quantitative ChIP-PCR analysis of PAX5 occupancy of the GRHL1 and GRHL3 regulatory regions was performed in HEK293 cells transfected with pcDNA3.1-N-DYK-PAX5. KRAS was used to identify non-specific interactions. Chromatin was immunoprecipitated with anti-DYK (FLAG) antibody or nonspecific antibody. The amount of DNA amplified from immunoprecipitated DNA was normalized to that amplified from input DNA. Data are shown as means ± SEM of experiments independently performed in triplicate, **significantly different at p≤ 0.01. (B) EMSA analysis performed with probes including PAX5 binding sequences: left panel–in the region -147/-128 (lane 2) of the GRHL1 promoter; right panel–in the region +3386/+3405 (lane 2) of the GRHL3 enhancer. Lane 1: probe only. Cold probe: unlabeled double-stranded oligonucleotides including -147/-128 (left panel, lane 3) or +3386/-3405 (right panel, lane 3) regions of GRHL genes (100-fold molar excess of competitors). Where indicated, 1 μg anti-PAX5 antibody was added per lane (ab15164, Abcam).
Fig 4.
Overexpression of KLF4 or PAX5 alters mRNA levels of GRHL genes.
(A-B) The mRNA expression levels of GRHL1, GRHL2 and GRHL3 genes in HEK293 cells transiently overexpressing KLF4 (A) or PAX5 (B). The results represent relative expression of the respective target gene vs HPRT gene. Data are shown as means ± SEM of experiments independently performed in triplicate, *significantly different at p≤ 0.05, **significantly different at p≤ 0.01.
Fig 5.
KLF4 changes the activity of GRHL regulatory regions.
(A) HEK293 cells were transfected with pcDNA3.1-KLF4 plasmid, pGL3-promoter vector containing the luciferase gene under the control of the fragment of the GRHL1 or GRHL2 or GRHL3 regulatory sequence with or without binding sites for KLF4 (named: KLF4/GRHL1-3-luc or NoKLF4/GRHL1-2-luc) and pRL-CMV Renilla luciferase control reporter vector. Data are shown as means ± SEM of experiments independently performed in triplicate, *significantly different at p≤ 0.05, **significantly different at p≤ 0.01. (B) Schematic representation of locations of the KLF4 binding site sequences in the promoters of GRHL1, GRHL2 and GRHL3 genes cloned into luciferase vectors.
Fig 6.
PAX5 changes the activity of GRHL regulatory regions.
(A) HEK293 cells were transfected with pcDNA3.1-N-DYK-PAX5 plasmid, pGL3-promoter vector containing the luciferase gene under the control of the fragment of the GRHL1 or GRHL3 regulatory sequence with or without binding sites for PAX5 (named: PAX5/GRHL1-luc or PAX5/GRHL3-luc or NoPAX5/GRHL1-luc or NoPAX5/GRHL3-luc and pRL-CMV Renilla luciferase control reporter vector. Data are shown as means ± SEM of experiments independently performed in triplicate, **significantly different at p≤ 0.01. (B) Schematic representation of locations of the PAX5 binding site sequences in the promoter of GRHL1 gene and in the enhancer of GRHL3 gene cloned into luciferase vectors.
Fig 7.
Impact of the presence of the minor frequency allele of SNP rs115898376 in the promoter of GRHL1 gene on KLF4 binding.
HEK293 cells were transfected with pcDNA3.1-KLF4 or pcDNA3.1-empty plasmid, pGL3-promoter vector containing the luciferase gene under the control of the fragment of the GRHL1 regulatory sequence with either the major or the minor frequency allele of SNP rs115898376 (named KLF4/GRHL-luc2 and KLF4/GRHL1-SNP-luc2), and pRL-CMV Renilla luciferase control reporter vector. The presence of the minor frequency allele of SNP rs115898376 in the promoter of GRHL1 gene alters the functioning of the KLF4 binding site.