9 ANALISIS
9.4 Objetivo específico 1
Differential chromatin opening makes the chromatin more accessible in some genomic regions rather than in others and therefore more accessible to the binding of specific TFs, likely involved in the regulation of nearby gene expression. Hence, de novo binding motif enrichment analysis, using Hypergeometric Optimization of Motif EnRichment (HOMER), was performed on statistically significant opening and closing ATAC-seq peaks (Bonferroni adjusted
p < 0.05) to identify the overepresented binding motifs and putative TFs that underly changes in gene expression upon decidualization.
A total of 17 motifs, named differentiated motifs 1-17 (DiffM1-17), were significantly enriched in 1,225 differential ATAC-seq peaks that significantly open upon decidualization and 7 motifs, named undifferentiated motifs 1-7 (UndiffM1- 7), were significantly overepresented in 278 closing ATAC-seq peaks (Figure 3.6). Data showed that closing or opening of genomic regions could also be predicted by the occurrence of the motifs (Figure 3.7). Within regions of chromatin opening, TF occupancy should result in a footprint. To investigate if the identified motifs were indicative of TF binding, ATAC-seq signal was averaged over all expected locations of the identified conserved motif sequences. Average ATAC- seq signal profile centered at each motif revealed sequence footprint reflecting factor occupancy (Figure 3.8). DNA-protein binding makes the occupied genomic region refractory to transposition, causing enrichment of positive strand reads 5’ of the binding motif paired with excess of negative strand reads 3’ of the motifs. Evidence of footprints revealed the presence of DNA-binding protein in all the conserved motifs except for Opening motif 10 and Closing motif 2. Absence of footprint at these genomic sites suggested that there was no evidence of TF occupancy. However, these chromatin regions might still play a role in the regulation of gene expression. For example, they might be involved in the folding of the chromatin to facilitate interactions between functionally related genes spatially separated along the genome.
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Next, to identify the putative TFs that might bind at these genomic locations, the short sequence motifs were matched against known TF datasets and their expression level was examined. HOMER motif analysis revealed overepresentation of binding sites representing high affinity binding motifs for known decidual TFs, including CCAAT/enhancer binding protein beta and delta (CEBPB/CEBPD), Fos-like antigen (FOSL2 or FRA2), forkhead box O1 (FOXO1), progesterone receptor (PGR), and signal transducer and activator of transcription 3 and 5 (STAT3/STAT5) (Mazur et al., 2015; Kaya et al., 2015; Jiang
et al., 2015; Kim et al., 2005). Depletion of TEA domain transcription factor 1 (TEAD1) binding motif was dectected upon decidualization (Figure 3.9). Interestingly, TEAD1 was previously shown to negatively regulate the expression of PRL, a well-established decidual marker (Kessler et al., 2008). A full list of the best match of TFs for all of the binding motifs enriched in the opening and closing motifs are shown in Appendix 1 and 2, respectively.
Cross-referencing the resulting ATAC-seq regions with available chromatin immunoprecipitation followed by sequencing (ChIP-seq) data (e.g. ENCODE database), for PGR, FOXO1 and FOSL2 in EnSCs, provided confidence in the identified binding domains (Vasquez et al., 2015). In agreement with their key role in decidualization, ChIP-seq data revealed that FOXO1 and PGR binding sites were overepresented in open chromatin regions. Particularly, chromatin regions enriched with both PGR and FOSL2 binding motifs were associated to open chromatin in decidualized EnSCs, whereas in absence of PGR binding site, FOSL2 binding domain was enriched in closing chromatin regions (Figure 3.10). Taken together, data showed that ATAC-seq accurately mapped dynamic changes upon decidual transformation. It identified binding sites for TFs known to play an essential role in promoting decidualization. However, ATAC-seq yielded novel candidate TFs likely to be involved in licensing genomic regions for remodelling in response to decidual transformation. The role of these putative novel transcriptional regulators in decidual transformation of EnSCs requires further investigations. Experimentally, RNA-seq or RT-qPCR and western blot could be applied to validate the induction of TFs of interest at a transcript and protein level. ChIP-qRT-PCR or cross-referencing with available ChIP-seq data
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(e.g. ENCODE database) would provide confidence in the transcriptional regulation of genes of interest by specific TFs. Furthermore, silencing of the most highly ranked conserved TFs by siRNA-mediated gene silencing could be performed to validate their role during the decidual process. In this work, RNA- seq was performed to examine gene expression of new putative decidual TFs. However, time limitations prevented me of persuing this line of investigation even further.
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Figure 3.6. Enriched TF binding motifs in opening and closing chromatin regions. De novo short sequence binding motif enrichment analysis revealed overrepresentation of 17 and 7 binding sites in statistically significant opening
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and closing ATAC-seq, respectively. The frequency (%) of peaks (blue bars) containing a given motif is shown relative to genomic regions randomly selected from the genome (orange bars) (±50 Kb from TSS, matching size, and GC/CpG content).
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Figure 3.7. Occurrence of TF binding motifs correlates to differential chromatin opening. The short sequence binding motifs resulted from HOMER motif analysis were associated with chromatin opening and closing over the whole ATAC-seq dataset. The frequency of opening and closing motifs (Y-axis) was identified in all ATAC-seq peaks. Peaks were sorted from most opening to most closing peak and ranks of peaks containing motifs were plotted as histograms (X-axis). ATAC-seq peaks containing Opening Motifs 1-17 are most frequently opening, whereas ATAC-seq peaks containing Closing Motifs 1-7 are more frequently closing. Empty bars indicate random expectation based on genomic background frequency.
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Figure 3.8. Footprint analysis. Footprint at the enriched motifs indicating evidence of TF occupancy. Graphs show average ATAC-seq signal profile centred at the opening (A) and closing (B) binding motifs, calculated within ±200 bp on enriched motif. Red and blue represent positive and negative strand cuts, respectively. Reduced read numbers in the region of the motif together with increase in positive and negative strand reads at 5’ and 3’ of the motif indicate enrichment of fragments that span the uncut (protected) region of DNA binding.
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Figure 3.9. Top 5 enriched and depleted binding motifs and coupled high binding affinity TFs. Bar graphs showing top 5 binding motifs, enriched in the opening and closing ATAC-seq peaks, matched with the most plausible differentially expressed TFs, based on motif specificity. In the bar graphs, the frequency (%) of peaks (blue bars) containing the motif is shown relative to genomic regions randomly selected from the genome (orange bars) (±50 Kb from TSS, matching size, and GC/CpG content). P indicates the p of the short sequence binding motifs.
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Figure 3.10. Mapping of known FOXO1, PGR and FOSL2 binding domains in the differential ATAC-seq peaks in decidualized EnSCs. Cross-reference of ATAC-seq data with public ChIP-seq data for the indicated key transcription factors during decidualization, revealed enrichment of FOXO1 and PGR binding motifs in the opening genomic regions; FOSL2 binding motif was enriched in closing chromatin regions, whereas FOSL2 in the presence of PGR binding domain was associated to opening chromatin regions. Again, ATAC-seq peaks were ranked from the most strongly opening to the most strongly closing peak.
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