Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 2 de 2
Filter
Add more filters










Database
Language
Publication year range
1.
Sci Rep ; 12(1): 5573, 2022 04 02.
Article in English | MEDLINE | ID: mdl-35368039

ABSTRACT

It is critical to understand the molecular mechanisms governing the regulation of MITF, a lineage specific transcription factor in melanocytes and an oncogene in melanoma. We identified PPP6C, a serine/threonine phosphatase, as a key regulator of MITF in melanoma. PPP6C is the only recurrently mutated serine/threonine phosphatase across all human cancers identified in sequencing studies and the recurrent R264C mutation occurs exclusively in melanoma. Using a zebrafish developmental model system, we demonstrate that PPP6C expression disrupts melanocyte differentiation. Melanocyte disruption was rescued by engineering phosphomimetic mutations at serine residues on MITF. We developed an in vivo MITF promoter assay in zebrafish and studied the effects of PPP6C(R264C) on regulating MITF promoter activity. Expression of PPP6C(R264C) cooperated with oncogenic NRAS(Q61K) to accelerate melanoma initiation in zebrafish, consistent with a gain of function alteration. Using a human melanoma cell line, we examined the requirement for PPP6C in proliferation and MITF expression. We show that genetic inactivation of PPP6C increases MITF and target gene expression, decreases sensitivity to BRAF inhibition, and increases phosphorylated MITF in a BRAF(V600E) mutant melanoma cell line. Our data suggests that PPP6C may be a relevant drug target in melanoma and proposes a mechanism for its action.


Subject(s)
Melanoma , Microphthalmia-Associated Transcription Factor , Animals , Carcinogenesis/metabolism , Cell Line, Tumor , Melanocytes/metabolism , Melanoma/genetics , Melanoma/metabolism , Microphthalmia-Associated Transcription Factor/genetics , Microphthalmia-Associated Transcription Factor/metabolism , Phosphoprotein Phosphatases/metabolism , Zebrafish/metabolism
2.
Nucleic Acids Res ; 48(7): e38, 2020 04 17.
Article in English | MEDLINE | ID: mdl-32064511

ABSTRACT

CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.


Subject(s)
CRISPR-Associated Protein 9 , CRISPR-Cas Systems , Gene Editing , Recombinational DNA Repair , Animals , CRISPR-Associated Protein 9/genetics , Fluorescent Dyes , Green Fluorescent Proteins/genetics , INDEL Mutation , Indicators and Reagents , Melanocytes , Nitroreductases/genetics , RNA/chemistry , Templates, Genetic , Zebrafish/embryology , Zebrafish/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...