Eric Van Otterloo

A polymorphism in IRF4 affects human pigmentation through a tyrosinase-dependent MITF/TFAP2A pathway.

Sequence polymorphisms linked to human diseases and phenotypes in genome-wide association studies often affect noncoding regions. A SNP within an intron of the gene encoding Interferon Regulatory Factor 4 (IRF4), a transcription factor with no known role in melanocyte biology, is strongly associated with sensitivity of skin to sun exposure, freckles, blue eyes, and brown hair color. Here, we demonstrate that this SNP lies within an enhancer of IRF4 transcription in melanocytes. The allele associated with this pigmentation phenotype impairs binding of the TFAP2A transcription factor that, together with the melanocyte master regulator MITF, regulates activity of the enhancer. Assays in zebrafish and mice reveal that IRF4 cooperates with MITF to activate expression of Tyrosinase (TYR), an essential enzyme in melanin synthesis. Our findings provide a clear example of a noncoding polymorphism that affects a phenotype by modulating a developmental gene regulatory network.

Maternal Interferon Regulatory Factor 6 is required for the differentiation of primary superficial epithelia in Danio and Xenopus embryos

Early in the development of animal embryos, superficial cells of the blastula form a distinct lineage and adopt an epithelial morphology. In different animals, the fate of these primary superficial epithelial (PSE) cells varies, and it is unclear whether pathways governing segregation of blastomeres into the PSE lineage are conserved. Mutations in the gene encoding Interferon Regulatory Factor 6 (IRF6) are associated with syndromic and non-syndromic forms of cleft lip and palate, consistent with a role for Irf6 in development of oral epithelia, and mouse Irf6 targeted null mutant embryos display abnormal differentiation of oral epithelia and skin. In Danio rerio (zebrafish) and Xenopus laevis (African clawed frog) embryos, zygotic irf6 transcripts are present in many epithelial tissues including the presumptive PSE cells and maternal irf6 transcripts are present throughout all cells at the blastula stage. Injection of antisense oligonucleotides with ability to disrupt translation of irf6 transcripts caused little or no effect on development. By contrast, injection of RNA encoding a putative dominant negative Irf6 caused epiboly arrest, loss of gene expression characteristic of the EVL, and rupture of the embryo at late gastrula stage. The dominant negative Irf6 disrupted EVL gene expression in a cell autonomous fashion. These results suggest that Irf6 translated in the oocyte or unfertilized egg suffices for early development. Supporting the importance of maternal Irf6, we show that depletion of maternal irf6 transcripts in X. laevis embryos leads to gastrulation defects and rupture of the superficial epithelium. These experiments reveal a conserved role for maternally-encoded Irf6 in differentiation of a simple epithelium in X. laevis and D. rerio. This epithelium constitutes a novel model tissue in which to explore the Irf6 regulatory pathway.

Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma. DOI: http://dx.doi.org/10.7554/eLife.06857.001

Novel Tfap2-mediated control of soxE expression facilitated the evolutionary emergence of the neural crest

Gene duplication has been proposed to drive the evolution of novel morphologies. After gene duplication, it is unclear whether changes in the resulting paralogs’ coding-regions, or in their cis-regulatory elements, contribute most significantly to the assembly of novel gene regulatory networks. The Transcription Factor Activator Protein 2 (Tfap2) was duplicated in the chordate lineage and is essential for development of the neural crest, a tissue that emerged with vertebrates. Using a tfap2-depleted zebrafish background, we test the ability of available gnathostome, agnathan, cephalochordate and insect tfap2 paralogs to drive neural crest development. With the exception of tfap2d (lamprey and zebrafish), all are able to do so. Together with expression analyses, these results indicate that sub-functionalization has occurred among Tfap2 paralogs, but that neo-functionalization of the Tfap2 protein did not drive the emergence of the neural crest. We investigate whether acquisition of novel target genes for Tfap2 might have done so. We show that in neural crest cells Tfap2 directly activates expression of sox10, which encodes a transcription factor essential for neural crest development. The appearance of this regulatory interaction is likely to have coincided with that of the neural crest, because AP2 and SoxE are not co-expressed in amphioxus, and because neural crest enhancers are not detected proximal to amphioxus soxE. We find that sox10 has limited ability to restore the neural crest in Tfap2-deficient embryos. Together, these results show that mutations resulting in novel Tfap2-mediated regulation of sox10 and other targets contributed to the evolution of the neural crest.

Differentiation of zebrafish melanophores depends on transcription factors AP2 alpha and AP2 epsilon.

A model of the gene-regulatory-network (GRN), governing growth, survival, and differentiation of melanocytes, has emerged from studies of mouse coat color mutants and melanoma cell lines. In this model, Transcription Factor Activator Protein 2 alpha (TFAP2A) contributes to melanocyte development by activating expression of the gene encoding the receptor tyrosine kinase Kit. Next, ligand-bound Kit stimulates a pathway activating transcription factor Microphthalmia (Mitf), which promotes differentiation and survival of melanocytes by activating expression of Tyrosinase family members, Bcl2, and other genes. The model predicts that in both Tfap2a and Kit null mutants there will be a phenotype of reduced melanocytes and that, because Tfap2a acts upstream of Kit, this phenotype will be more severe, or at least as severe as, in Tfap2a null mutants in comparison to Kit null mutants. Unexpectedly, this is not the case in zebrafish or mouse. Because many Tfap2 family members have identical DNA–binding specificity, we reasoned that another Tfap2 family member may work redundantly with Tfap2a in promoting Kit expression. We report that tfap2e is expressed in melanoblasts and melanophores in zebrafish embryos and that its orthologue, TFAP2E, is expressed in human melanocytes. We provide evidence that Tfap2e functions redundantly with Tfap2a to maintain kita expression in zebrafish embryonic melanophores. Further, we show that, in contrast to in kita mutants where embryonic melanophores appear to differentiate normally, in tfap2a/e doubly-deficient embryonic melanophores are small and under-melanized, although they retain expression of mitfa. Interestingly, forcing expression of mitfa in tfap2a/e doubly-deficient embryos partially restores melanophore differentiation. These findings reveal that Tfap2 activity, mediated redundantly by Tfap2a and Tfap2e, promotes melanophore differentiation in parallel with Mitf by an effector other than Kit. This work illustrates how analysis of single-gene mutants may fail to identify steps in a GRN that are affected by the redundant activity of related proteins.

New Functional Signatures for Understanding Melanoma Biology from Tumor Cell Lineage-Specific Analysis

SUMMARY Molecular signatures specific to particular tumor types are required to design treatments for resistant tumors. However, it remains unclear whether tumors and corresponding cell lines used for drug development share such signatures. We developed similarity core analysis (SCA), a universal and unsupervised computational framework for extracting core molecular features common to tumors and cell lines. We applied SCA to mRNA/miRNA expression data from various sources, comparing melanoma cell lines and metastases. The signature obtained was associated with phenotypic characteristics in vitro, and the core genes CAPN3 and TRIM63 were implicated in melanoma cell migration/invasion. About 90% of the melanoma signature genes belong to an intrinsic network of transcription factors governing neural development (TFAP2A, DLX2, ALX1, MITF, PAX3, SOX10, LEF1, and GAS7) and miRNAs (211-5p, 221-3p, and 10a-5p). The SCA signature effectively discriminated between two subpopulations of melanoma patients differing in overall survival, and classified MEKi/BRAFi-resistant and -sensitive melanoma cell lines.

Gene regulatory evolution and the origin of macroevolutionary novelties: Insights from the neural crest

The appearance of novel anatomic structures during evolution is driven by changes to the networks of transcription factors, signaling pathways, and downstream effector genes controlling development. The nature of the changes to these developmental gene regulatory networks (GRNs) is poorly understood. A striking test case is the evolution of the GRN controlling development of the neural crest (NC). NC cells emerge from the neural plate border (NPB) and contribute to multiple adult structures. While all chordates have a NPB, only in vertebrates do NPB cells express all the genes constituting the neural crest GRN (NC‐GRN). Interestingly, invertebrate chordates express orthologs of NC‐GRN components in other tissues, revealing that during vertebrate evolution new regulatory connections emerged between transcription factors primitively expressed in the NPB and genes primitively expressed in other tissues. Such interactions could have evolved by two mechanisms. First, transcription factors primitively expressed in the NPB may have evolved new DNA and/or cofactor binding properties (protein neofunctionalization). Alternately, cis‐regulatory elements driving NPB expression may have evolved near genes primitively expressed in other tissues (cis‐regulatory neofunctionalization). Here we discuss how gene duplication can, in principle, promote either form of neofunctionalization. We review recent published examples of interspecies gene‐swap, or regulatory‐element‐swap, experiments that test both models. Such experiments have yielded little evidence to support the importance of protein neofunctionalization in the emergence of the NC‐GRN, but do support the importance of novel cis‐regulatory elements in this process. The NC‐GRN is an excellent model for the study of gene regulatory and macroevolutionary innovation. genesis 51:457–470.

TFAP2 paralogs regulate melanocyte differentiation in parallel with MITF

Mutations in the gene encoding transcription factor TFAP2A result in pigmentation anomalies in model organisms and premature hair graying in humans. However, the pleiotropic functions of TFAP2A and its redundantly-acting paralogs have made the precise contribution of TFAP2-type activity to melanocyte differentiation unclear. Defining this contribution may help to explain why TFAP2A expression is reduced in advanced-stage melanoma compared to benign nevi. To identify genes with TFAP2A-dependent expression in melanocytes, we profile zebrafish tissue and mouse melanocytes deficient in Tfap2a, and find that expression of a small subset of genes underlying pigmentation phenotypes is TFAP2A-dependent, including Dct, Mc1r, Mlph, and Pmel. We then conduct TFAP2A ChIP-seq in mouse and human melanocytes and find that a much larger subset of pigmentation genes is associated with active regulatory elements bound by TFAP2A. These elements are also frequently bound by MITF, which is considered the “master regulator” of melanocyte development. For example, the promoter of TRPM1 is bound by both TFAP2A and MITF, and we show that the activity of a minimal TRPM1 promoter is lost upon deletion of the TFAP2A binding sites. However, the expression of Trpm1 is not TFAP2A-dependent, implying that additional TFAP2 paralogs function redundantly to drive melanocyte differentiation, which is consistent with previous results from zebrafish. Paralogs Tfap2a and Tfap2b are both expressed in mouse melanocytes, and we show that mouse embryos with Wnt1-Cre-mediated deletion of Tfap2a and Tfap2b in the neural crest almost completely lack melanocytes but retain neural crest-derived sensory ganglia. These results suggest that TFAP2 paralogs, like MITF, are also necessary for induction of the melanocyte lineage. Finally, we observe a genetic interaction between tfap2a and mitfa in zebrafish, but find that artificially elevating expression of tfap2a does not increase levels of melanin in mitfa hypomorphic or loss-of-function mutants. Collectively, these results show that TFAP2 paralogs, operating alongside lineage-specific transcription factors such as MITF, directly regulate effectors of terminal differentiation in melanocytes. In addition, they suggest that TFAP2A activity, like MITF activity, has the potential to modulate the phenotype of melanoma cells.

MEMO1 drives cranial endochondral ossification and palatogenesis

The cranial base is a component of the neurocranium and has a central role in the structural integration of the face, brain and vertebral column. Consequently, alteration in the shape of the human cranial base has been intimately linked with primate evolution and defective development is associated with numerous human facial abnormalities. Here we describe a novel recessive mutant mouse strain that presented with a domed head and fully penetrant cleft secondary palate coupled with defects in the formation of the underlying cranial base. Mapping and non-complementation studies revealed a specific mutation in Memo1 - a gene originally associated with cell migration. Expression analysis of Memo1 identified robust expression in the perichondrium and periosteum of the developing cranial base, but only modest expression in the palatal shelves. Fittingly, although the palatal shelves failed to elevate in Memo1 mutants, expression changes were modest within the shelves themselves. In contrast, the cranial base, which forms via endochondral ossification had major reductions in the expression of genes responsible for bone formation, notably matrix metalloproteinases and markers of the osteoblast lineage, mirrored by an increase in markers of cartilage and extracellular matrix development. Concomitant with these changes, mutant cranial bases showed an increased zone of hypertrophic chondrocytes accompanied by a reduction in both vascular invasion and mineralization. Finally, neural crest cell-specific deletion of Memo1 caused a failure of anterior cranial base ossification indicating a cell autonomous role for MEMO1 in the development of these neural crest cell derived structures. However, palate formation was largely normal in these conditional mutants, suggesting a non-autonomous role for MEMO1 in palatal closure. Overall, these findings assign a new function to MEMO1 in driving endochondral ossification in the cranium, and also link abnormal development of the cranial base with more widespread effects on craniofacial shape relevant to human craniofacial dysmorphology.

Beyond MITF: Multiple transcription factors directly regulate the cellular phenotype in melanocytes and melanoma

MITF governs multiple steps in the development of melanocytes, including specification from neural crest, growth, survival, and terminal differentiation. In addition, the level of MITF activity determines the phenotype adopted by melanoma cells, whether invasive, proliferative, or differentiated. However, MITF does not act alone. Here, we review literature on the transcription factors that co‐regulate MITF‐dependent genes. ChIP‐seq studies have indicated that the transcription factors SOX10, YY1, and TFAP2A co‐occupy subsets of regulatory elements bound by MITF in melanocytes. Analyses at single loci also support roles for LEF1, RB1, IRF4, and PAX3 acting in combination with MITF, while sequence motif analyses suggest that additional transcription factors colocalize with MITF at many melanocyte‐specific regulatory elements. However, the precise biochemical functions of each of these MITF collaborators and their contributions to gene expression remain to be elucidated. Analogous to the transcriptional networks in morphogen‐patterned tissues during embryogenesis, we anticipate that the level of MITF activity is controlled not only by the concentration of activated MITF, but also by additional transcription factors that either quantitatively or qualitatively influence the expression of MITF‐target genes.