Combinatorial recruitment of CREB, C/EBPβ and c-Jun determines activation of promoters upon keratinocyte differentiation

Abstract

Background: Transcription factors CREB, C/EBPβ and Jun regulate genes involved in keratinocyte proliferation and differentiation. We questioned if specific combinations of CREB, C/EBPβ and c-Jun bound to promoters correlate with RNA polymerase II binding, mRNA transcript levels and methylation of promoters in proliferating and differentiating keratinocytes.

Results: Induction of mRNA and RNA polymerase II by differentiation is highest when promoters are bound by C/EBP β alone, C/EBPβ together with c-Jun, or by CREB, C/EBPβ and c-Jun, although in this case CREB binds with low affinity. In contrast, RNA polymerase II binding and mRNA levels change the least upon differentiation when promoters are bound by CREB either alone or in combination with C/EBPβ or c-Jun. Notably, promoters bound by CREB have relatively high levels of RNA polymerase II binding irrespective of differentiation. Inhibition of C/EBPβ or c-Jun preferentially represses mRNA when gene promoters are bound by corresponding transcription factors and not CREB. Methylated promoters have relatively low CREB binding and, accordingly, those which are bound by C/EBPβ are induced by differentiation irrespective of CREB. Composite "Half and Half" consensus motifs and co localizing consensus DNA binding motifs are overrepresented in promoters bound by the combination of corresponding transcription factors.

Conclusion: Correlational and functional data describes combinatorial mechanisms regulating the activation of promoters. Colocalization of C/EBPβ and c-Jun on promoters without strong CREB binding determines high probability of activation upon keratinocyte differentiation.

Figure 1. Upon keratinocyte differentiation RNAP binding and mRNA levels are preferentially induced when promoters are bound by combinations of C/EBPβ and cJun.

A. Scatterplott of changes in RNAP binding after differentiation versus RNAP binding in undifferentiated keratinocytes show 797 and 841 promoters are induced or repressed upon differentiation. These changes correlate with changes in mRNA levels. B. Transcription factors CREB, C/EBPβ and c-Jun bind distinct set of promoters in differentiated keratinocytes. Euler diagrams show that promoters bound by C/EBPβ and c-Jun are preferentially induced by differentiation and promoters bond by CREB are not. C. Fraction of promoters bound by different combination of transcription factors in differentiated keratinocytes where RNAP binding is repressed (white bars) or induced by differentiation (black bars). D. Fraction of genes with mRNA levels induced or repressed by differentiation more than 1.4 times in groups of promoters bound by different combinations of transcription factors. * - values are different from expected (p<0.005 using a two-tailed unpaired t-test).

Figure 2. Colocalization of C/EBPβ or c-Jun with CREB determine genes which expression and induction upon differentiation is dependent on C/EBPβ and c-Jun functions.

A. Fraction of genes repressed by A-C/EBP (left panel) or A-Fos (right panel) in differentiated keratinocytes in groups of promoters bound by different combinations of transcription factors. B. Fraction of genes induced by differentiation and repressed by A-C/EBP (left panel) or A-Fos (right panel) in differentiated keratinocytes in groups of genes induced by differentiation which promoters are bound by different combinations of transcription factors. * - numbers are different from expected (p<0.05 using a two-tailed unpaired t-test).

Figure 3. CREB binding is relatively low in the group of promoters bound by CREB, C/EBPβ and cJun and induced by differentiation.

A. Transcription factor binding distributions in all promoters, promoters bound by CREB, C/EBPβ and c-Jun, and promoters bound by all three of these proteins that are also either induced or repressed upon differentiation. Columns show the mean value of the binding affinity for each of the three transcription factors in these four groups of promoters, while error bars show the 15% and 85% percentiles. The binding affinity of CREB is significantly lower in promoters bound by all three transcription factors and induced by differentiation. Number on top represents the p-value from an unpaired t-test. B. Chip-PCR for promoter regions of SPRP1A and DNAse1L3 induced by differentiation. CREB binding is low in comparison with C/EBPβ and c-Jun. 3′ GAPDH region was not enriched in these samples.

Figure 4. Colocalization of C/EBPβ and c-Jun with CREB is associated with high probability of RNAP binding in all promoters and promoters induced by differentiation.

A. Scatter plots of transcription factors and RNAP binding show that 95% of promoters bound by CREB, 82% of C/EBPβ and 62% of c-Jun bound promoters are also bound by RNAP. Lines are RNAP binding thresholds. B. RNAP binding percentiles (15%, 50% and 85%) in promoters bound by different combinations of transcription factors in differentiated keratinocytes. C. RNAP binding percentiles (15%, 50% and 85%) in promoters induced by differentiation and bound by different combinations of transcription factors. Numbers over the bars represent t-test values. Dotted lines are RNAP binding thresholds.

Figure 5. C/EBPβ preferentially binds to methylated promoters and methylated promoters bound by C/EBPβ are preferentially induced by differentiation.

A. Euler diagrams of methylated and unmethylated promoters bound by different combination of transcription factors show that CREB binding is depleted on methylated promoters while C/EBPβ and c-Jun binding is overrepresented. B. Percent of promoters with RNAP is induced or repressed by differentiation in promoters bound by different combinations of transcription factors in differentiated keratinocytes for unmethylated (left) and methylated promoters (right). C. Percent of genes with mRNA is induced or repressed by differentiation in promoters bound by different combinations of transcription factors in differentiated keratinocytes for unmethylated (left) and methylated promoters (right). * - numbers are significantly different from expected, (p<0.05).

Figure 6. Combinations of consensus motifs and composite motifs are enriched in the groups of promoters bound by different combinations of transcription factors.

A. Top DNA motifs mostly enriched in test sets v.s. background sets of promoters in the −500 bp…0 bp relative to the transcription start site. Motifs are sorted by enrichment and statistical confidence level using CisFinder. For CREB, c-Jun C/EBPβ set cJun and C/EBPβ bound promoters were used as a background. B. Enrichment of promoters containing only two or three transcription factors consensus binding motifs in groups of promoters bound by different combination of transcription factors in undifferentiated keratinocytes. Consensus motifs for CREB (CRE) - TGACGTCA, C/EBPβ - (C/EBP) TTGCGCAA and for c-Jun (AP-1) TGA(C/G)TCA. Note that promoters containing combination of motifs are overrepresented in the groups of promoters bound by corresponding transcription factor. * - numbers are different from expected (p<0.05).