Year: 2017
Type: article
Abstract: Vertebrate pigmentation is a process directed by various transcriptional regulators that drive the specification of melanoblasts from neural crest cells, their proliferation and migration, and terminal differentiation into melanocytes, culminating in the generation of pigment and its distribution to surrounding keratinocytes. The transcription factor MITF has been coined the “master regulator” of melanocyte development (reviewed in (Steingrímsson, Copeland, & Jenkins, 2004)), as it drives both the specification and differentiation of melanocytes. It is associated with pigmentation disorders in human, and Mitf mutant mice have a varying degree of pigmentation defects. Several other transcription factors have been shown to act in the melanocyte gene regulatory network. Notably, mutations in TFAP2A are associated with premature hair greying in humans, and animals carrying a neural crest-specific Tfap2a deletion in mice have a white belly spot. In a recent paper in PLOS Genetics, Seberg et al. elegantly dissect the molecular mechanism of TFAP2 transcription factor paralogs in melanocyte specification and differentiation. Using mouse and human cells together with mouse and zebrafish animal models, they show that the TFAP2 factors act in parallel with the previously coined “master regulator” of melanogenesis MITF. The authors start by analyzing tfap2a−/− mutant zebrafish embryos. Previously these had been shown to have about a 30% reduction in melanocytes which also showed defective migration. Here, they demonstrate that acquisition of pigmentation in melanocytes is delayed in mutants in comparison with wild type. A global gene expression analysis revealed over two thousand tfap2a-dependent differentially expressed genes between wild type and mutant fish trunks. These included more than half of a set of previously defined zebrafish pigmentation genes that showed decreased expression levels in the mutant fish, implying that tfap2a significantly impacts the regulation of pigmentation genes in zebrafish. To provide increased melanocyte lineage specificity to their analyses, they performed a global transcriptional analysis in immortalized mouse melanocytes upon Tfap2a knockdown. This revealed that a subset of mouse melanocyte differentiation genes overlapping that of differentially regulated orthologs in zebrafish, also failed to be induced. However, the number of differentially expressed genes upon Tfap2a knockdown in mouse melanocytes was perhaps surprisingly small. Interestingly, the expression of Mitf orthologs was not altered in either zebrafish or mouse, implying that their expression is TFAP2A independent. In an effort to define the melanocyte-specific binding targets of TFAP2A, Seberg et al. characterized the genomewide occupancy of TFAP2A in both mouse and human melanocytes. These analyses showed that both in mouse and in human melanocytes, a more proximal binding of TFAP2A to gene promoters correlated with increased expression levels, underpinning the notion that TFAP2A might be an important positive driver of gene expression in melanocytes. Interestingly, they found a high concordance between orthologous gene targets in mouse and human melanocytes, despite a clear divergence between the position of the bound cis-regulatory elements (CREs), such as enhancers. This implies that the distribution of CREs is altered in parallel with the acquisition of novel binding sites of TFAP2A during evolution. Indeed, in a study on cis-regulatory divergence in human vs. chimp neural crest, Prescott et al. found that CRE divergence was linked to quantitative expression differences of neural crest regulators and that one of the motifs associated with the largest species bias in the location of active CREs was the TFAP2A motif, where mutations in the motif correlated with the loss of CREs, and the gain of a motif associated with novel CREs (Prescott et al., 2015). ‘a large subset of actively transcribed melanocyte genes are co-regulated by TFAP2A and MITF’ Comparing the TFAP2A peaks to published binding profiles of the factor in kidney and epididymis, Seberg et al. show that about two-thirds of the binding events are unique in melanocytes and are highly enriched for the binding motifs of transcription factors with a known role in melanocytes, including that of SOX10 as well as MITF. The high enrichment of the melanocyte-specific MITF M-box on TFAP2A binding sites further strengthens the case for TFAP2A specifically regulating melanocyte gene expression. Indeed, comparing MITF and TFAP2A binding sites in human melanocytes, using a previously published data set for MITF, revealed an extensive overlap in binding events between the two factors (39%) with an even greater proportion (77%) of shared target genes when restricting the analysis to binding events occurring in active chromatin. As with many transcription factors, TFAP2A can both repress and activate transcription. To further functionally dissect TFAP2A and MITF binding sites, the authors were able to divide them into two groups based on the density of the histone-3 lysine-27 acetylation (H3K27ac) tags marking active CREs, obtained from another published study of human melanocytes. This functional dissection of binding events revealed that the great majority, or 76% of the co-occupied TFAP2A and MITF peaks, were on active CREs, strongly arguing the case that a large subset of actively transcribed melanocyte genes are coregulated by the two factors. As enhancer elements are considered to be drivers of tissue-specific gene expression (Visel et al., 2009), the authors compared their TFAP2A binding data to a published data set of immortalized mouse melanocyte enhancers. This showed that the majority of melanocyte enhancers (70%) are bound by TFAP2A. It is interesting to compare these data to another analysis of TFAP2A occupancy on enhancers, namely in neural crest precursors derived from human embryonic stem cell cultures (Rada-Iglesias et al., 2012). In the neural crest precursor cells, TFAP2A still occupies a significant proportion of enhancers, or 39%. However, this is a significantly lower proportion of enhancers compared to the melanocyte enhancer occupancy of TFAP2A in the current study. Whether this reflects the different degree of differentiation of the two cell stages or perhaps a higher number of marked enhancers in the precursors is not clear. It would be interesting to compare enhancers from neural crest precursor cells to those of melanocytes, to determine to which extent TFAP2A is already bound to its prospective melanocyte enhancers in the precursor cells or whether the binding repertoire is altered during differentiation by the redistribution of TFAP2A to previously non-bound CREs. The authors further deepen their analysis by looking at super-enhancers that tend to have highly active chromatin and are strongly associated with lineage-restricted gene expression. Seberg et al. used public data sets to define melanocyte-specific super-enhancers. Interestingly, the melanocyte-specific super-enhancers had a striking association of TFAP2A and MITF co-occupancy where over 80% of these highly active lineage specific drivers of gene expression are occupied by both factors, and the strongest scoring super-enhancers are co-occupied by both factors. Indeed, defined super-enhancers bound by neither factor tended to have a lower super-enhancer score. Such a high co-occupancy on super-enhancers argues strongly for a co-operative role of the two factors in melanocyte-specific gene regulation. However, despite the high degree of occupancy of TFAP2A on melanocyte-specific CREs, many of the genes associated with the peaks did not show TFAP2A-dependent expression, raising the issue of possible redundancy with other TFAP2 paralogs expressed in melanocytes. Using a transcriptional reporter the authors show that indeed, despite TRPM1 expression being TFAP2A independent, its promoter exhibited a dramatic reduction in activity upon deletion of TFAP2 binding motifs. The reduction in TRPM1 promoter activity upon TFAP2 motif deletion was even greater than upon deleting the MITF binding E-box, arguing for a strong influence of a redundant TFAP2 factor on TRPM1 transcription. To resolve this issue, in vivo Seberg et al. moved on to genetic studies in mice. They performed a melanocyte-specific conditional deletion of both Tfap2a and Tfap2b, the only other Tfap2 family member expressed in mouse melanocytes, to dissect their possible redundancy. Using Wnt1-Cre to conditionally delete floxed alleles of the two genes in the neural crest, they showed that single mutants of either gene did not exhibit a significant reduction in melanocyte number or differentiation at E12.0. However, simultaneous conditional mutation of Tfap2a and Tfap2b led to a significantly reduced melanocyte number. Importantly, they ruled out that the possibility that the defect was simply due to a migratory failure of melanoblasts, as the reduced cell number is apparent already in premigratory cells. Other neural crest derivatives are observed, however, also ruling out a general neural crest defect. These experiments solidly establish that melanocyte specification and differentiation depend on the redundant action of the TFAP2 transcription factor paralogs TFAP2A and TFAP2B. The high degree of co-occupancy of TFAP2A and MITF on CREs then leads the authors to hypothesize that the factors act in synergy to drive melanocyte-specific gene expression and use zebrafish to test that assumption. Indeed, mitfa:tfap2a double mutants show a greater-than-additive melanocyte phenotype, when mitfa null (mitfaw2) or mitfa hypomorphic (mitfaz25) alleles are crossed with tfap2a loss of function mutants, arguing for a synergistic mechanism of action. The authors had previously shown that a forced expression of mitfa in tfap2a/tfap2e-depleted zebrafish embryos was able to partially compensate for the melanocyte defect induced by the depletion. This implies that either MITF can be functionally redundant with the TFAP2 factors or that Mitfa induces tfap2 orthologues in melanocytes. The authors perform the converse experiment and attempt to rescue melanocytes in mitfa mutant zebrafish by the injection of constructs driving Tfap2a expression from the mitfa promoter. In this case, Tfap2a failed to rescue the melanocyte defect, in terms of both melanocyte number and degree of cellular pigmentation. The trivial explanation of this different ability of mitfa and tfap2a to reciprocally rescue one another's function could be that indeed mitfa drives tfap2a expression, as the experiment involved a knockdown mechanism rather than a genetic mutation. However, another possibility is that the two factors use different molecular mechanisms to positively influence transcriptional regulation. This could mean that an increased density of MITF on CREs such as super-enhancers is somehow more capable of overcoming the loss of TFAP2 factors, perhaps by increased recruitment of certain cofactors that are able to increase the rate of transcription in the absence of TFAP2 paralogs. Conversely, TFAP2 recruited cofactors might not have this ability to positively influence transcription in the absence of MITF. Another explanation mentioned by the authors could be that MITF acts as a pioneer factor in melanocytes, binding to previously inaccessible sites in chromatin, driving subsequent transcription factor recruitment to the sites. In that case, such sites normally cobound by TFAP2A/TFAP2B and MITF would be inaccessible when MITF is mutated, precluding the activation of these sites by the TFAP2 paralogs. Conversely, when the TFAP2 factors are mutated, MITF would already have opened up chromatin on CREs, and an increased binding frequency/density of MITF might overcome the loss of TFAP2 paralogs. This idea is particularly enticing considering the widespread TFAP2A binding and strong association with functional CREs in neural crest cells and that TFAP2A binding sites might need to be at least partially redistributed from neural crest targets to melanocyte target sites. Indeed, it will be interesting to follow future studies into the effect of TFAP2 family members as well as MITF on chromatin architecture and modification on CREs in the melanocyte lineage. The study described above is a careful dissection of the role of TFAP2 paralogs in melanocyte biology, where the authors creatively use the strengths of different model systems, as well as the integration of their genomewide assays with published data sets, to uncover the TFAP2 dependent mechanism of action, and integrate them firmly into the context of previous knowledge of transcriptional control of melanogenesis. One unavoidable question these analyses bring up is whether the concept of MITF as the master regulator of melanocyte biology can still hold up. The synergistic mechanism of action of MITF together with TFAP2 paralogs, including a large set of joint target genes, argues that the TFAP2 factors should be added to the repertoire of genes acting at the top of a hierarchical model of transcriptional gene regulation in melanocytes. However, as our general knowledge of gene regulatory networks expands, it is becoming evident that perhaps this hierarchical model of transcriptional regulation needs to be revised in the favor of a more complex model of parallel mechanisms of gene expression control.
Source: Pigment Cell & Melanoma Research
DOI: 10.1111/pcmr.12604
Author: Erna Magnúsdóttir
Language: English
Subfield: Cell Biology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2623979344