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Find video protocols related to scientific articles indexed in Pubmed.
The basic-helix-loop-helix-leucine zipper gene Mitf: analysis of alternative promoter choice and splicing.
Methods Mol. Biol.
PUBLISHED: 08-10-2010
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The activity of transcription factors is often regulated by Post-translational modifications. A precondition for such modifications is the presence, in the corresponding mRNAs, of the exons that either directly encode the modifiable residues in question, or encode protein domains that influence their modification indirectly. The inclusion or exclusion of coding exons is regulated predominantly by alternative splicing but can also depend on promoter choice and polyadenylation site selection. Information about exon inclusion and exclusion, both qualitatively and quantitatively, is particularly important for experiments designed to mutate endogenous codons because such mutations can alter splicing patterns. Therefore, we here describe methods employed to quantitate exon inclusion and exclusion, using as example a mouse transcription factor gene, Mitf.
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Allele-specific genetic interactions between Mitf and Kit affect melanocyte development.
Pigment Cell Melanoma Res
PUBLISHED: 03-29-2010
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The tyrosine kinase receptor KIT and the transcription factor MITF, each required for melanocyte development, have been shown to interact functionally both in vitro and in vivo. In vitro, KIT signaling leads to MITF phosphorylation, affecting MITF activity and stability. In vivo, the presence of the Mitf (Mi-wh) allele exacerbates the spotting phenotype associated with heterozygosity for Kit mutations. Here, we show that among a series of other Mitf alleles, only the recessive Mitf (mi-bws) mimics the effect of Mitf (Mi-wh) on Kit. Intriguingly, Mitf (mi-bws) is characterized by a splice defect that leads to a reduction of RNAs containing MITF exon 2B which encodes serine-73, a serine phosphorylated upon KIT signaling. Nevertheless, other Mitf alleles that generally affect Mitf RNA levels, or carry a serine-73-to-alanine mutation that specifically reduces exon 2B-containing RNAs, do not show similar interactions with Kit in vivo. We conclude that the recessive Mitf (mi-bws) is a complex allele that can display a semi-dominant effect when present in a Kit-sensitized background. We suggest that human disease variability may equally be due to complex, allele-specific interactions between different genes.
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ERK-regulated differential expression of the Mitf 6a/b splicing isoforms in melanoma.
Pigment Cell Melanoma Res
PUBLISHED: 11-06-2009
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The master regulator of the melanocyte lineage Mitf is intimately involved in development as well as melanoma, controlling cell survival, differentiation, proliferation and metastasis/migration. Consistent with its central role, Mitf expression and Mitf post-translational modifications are tightly regulated. An additional potential level of regulation is afforded by differential splicing of Mitf exon-6 leading to the generation of two isoforms that differ by the presence of six amino-acids in the Mitf (+) isoform and which have differential effects on cell cycle progression. However, whether the ratio of the two isoforms is regulated and whether their expression correlates with melanoma progression is not known. Here, we show that the differential expression of the Mitf 6a/b isoforms is dependent on the MAPKinase signalling, being linked to the activation of MEK1-ERK2, but not to N-RAS/B-RAF mutation status. In addition, quantification of Mitf 6a/b splicing forms in 86 melanoma samples revealed substantially increased levels of the Mitf (-) form in a subset of metastatic melanomas. The results suggest that differential expression of the Mitf 6a/b isoforms may represent an additional mechanism for regulating Mitf function and melanoma biology.
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The role of MITF phosphorylation sites during coat color and eye development in mice analyzed by bacterial artificial chromosome transgene rescue.
Genetics
PUBLISHED: 07-27-2009
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The microphthalmia-associated transcription factor (Mitf) has emerged as an important model for gene regulation in eukaryotic organisms. In vertebrates, it regulates the development of several cell types including melanocytes and has also been shown to play an important role in melanoma. In vitro, the activity of MITF is regulated by multiple signaling pathways, including the KITL/KIT/B-Raf pathway, which results in phosphorylation of MITF on serine residues 73 and 409. However, the precise role of signaling to MITF in vivo remains largely unknown. Here, we use a BAC transgene rescue approach to introduce specific mutations in MITF to study the importance of specific phospho-acceptor sites and protein domains. We show that mice that carry a BAC transgene where single-amino-acid substitutions have been made in the Mitf gene rescue the phenotype of the loss-of-function mutations in Mitf. This may indicate that signaling from KIT to MITF affects other phospho-acceptor sites in MITF or that alternative sites can be phosphorylated when Ser73 and Ser409 have been mutated. Our results have implications for understanding signaling to transcription factors. Furthermore, as MITF and signaling mechanisms have been shown to play an important role in melanomas, our findings may lead to novel insights into this resilient disease.
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On the interplay of telomeres, nevi and the risk of melanoma.
PLoS ONE
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The relationship between telomeres, nevi and melanoma is complex. Shorter telomeres have been found to be associated with many cancers and with number of nevi, a known risk factor for melanoma. However, shorter telomeres have also been found to decrease melanoma risk. We performed a systematic analysis of telomere-related genes and tagSNPs within these genes, in relation to the risk of melanoma, dysplastic nevi, and nevus count combining data from four studies conducted in Italy. In addition, we examined whether telomere length measured in peripheral blood leukocytes is related to the risk of melanoma, dysplastic nevi, number of nevi, or telomere-related SNPs. A total of 796 cases and 770 controls were genotyped for 517 SNPs in 39 telomere-related genes genotyped with a custom-made array. Replication of the top SNPs was conducted in two American populations consisting of 488 subjects from 53 melanoma-prone families and 1,086 cases and 1,024 controls from a case-control study. We estimated odds ratios for associations with SNPs and combined SNP P-values to compute gene region-specific, functional group-specific, and overall P-value using an adaptive rank-truncated product algorithm. In the Mediterranean population, we found suggestive evidence that RECQL4, a gene involved in genome stability, RTEL1, a gene regulating telomere elongation, and TERF2, a gene implicated in the protection of telomeres, were associated with melanoma, the presence of dysplastic nevi and number of nevi, respectively. However, these associations were not found in the American samples, suggesting variable melanoma susceptibility for these genes across populations or chance findings in our discovery sample. Larger studies across different populations are necessary to clarify these associations.
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In vivo role of alternative splicing and serine phosphorylation of the microphthalmia-associated transcription factor.
Genetics
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The microphthalmia-associated transcription factor (MITF) is a basic helix-loop-helix leucine zipper protein that plays major roles in the development and physiology of vertebrate melanocytes and melanoma cells. It is regulated by post-translational modifications, including phosphorylation at serine 73, which based on in vitro experiments imparts on MITF an increased transcriptional activity paired with a decreased stability. Serine 73 is encoded by the alternatively spliced exon 2B, which is preferentially skipped in mice carrying a targeted serine-73-to-alanine mutation. Here, we measured the relative abundance of exon 2B+ and exon 2B- RNAs in freshly isolated and FACS-sorted wild-type melanoblasts and melanocytes and generated a series of knock-in mice allowing forced incorporation of either alanine, aspartate, or wild-type serine at position 73. None of these knock-in alleles, however, creates a striking pigmentation phenotype on its own, but differences between them can be revealed either by a general reduction of Mitf transcript levels or in heteroallelic combinations with extant Mitf mutations. In fact, compared with straight serine-73 knock-in mice with their relative reduction of 2B+ Mitf, forced incorporation of alanine 73 leads to greater increases in MITF protein levels, melanoblast and melanocyte numbers, and extent of pigmentation in particular allelic combinations. These results underscore, in vivo, the importance of the link between alternative splicing and post-translational modifications and may bear on the recent observation that exon 2B skipping can be found in metastatic melanoma.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.