Expression data from non-pigmented and pigmented mouse melanocytes
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ABSTRACT: Immortalized, amelanotic melanocytes isolted from skin of Balb/c express enzymatically-inactive tyrosinase due to a homozygous point mutation (TGT->TCT) in tyrosinase gene, resulting in a lack of melanin . To serve as a control cell line, pigmentation was restored in these cells by correcting the point mutation using an RNA-DNA oligonucleotide (kingly gift from Dr. Alexeev Y. Vitali). We used microarray to detail the effect of tyrosinase mutation on gene expression in normal versus mutant melanocytes. Pigmented and non-pigmented melanocytes were grown for RNA extraction and hybridization on Affymetrix microarrays. We used both normal and mutant melanocyte in order to obtain expression profiles. We then examined variances in gene expression between the two cell lines.
Project description:Immortalized, amelanotic melanocytes isolted from skin of Balb/c express enzymatically-inactive tyrosinase due to a homozygous point mutation (TGT->TCT) in tyrosinase gene, resulting in a lack of melanin . To serve as a control cell line, pigmentation was restored in these cells by correcting the point mutation using an RNA-DNA oligonucleotide (kingly gift from Dr. Alexeev Y. Vitali). We used microarray to detail the effect of tyrosinase mutation on gene expression in normal versus mutant melanocytes.
Project description:The hair follicle bulb is the only site of pigment production for the hair shaft and melanogenically active melanocytes are located in the upper hair matrix. We conducted a microarray study to discover gene expression patterns that may be implicated in the lack of melanogenesis in gray hair follicles (HF). Pigmented and non-pigmented HFs collected from the same individuals were micro-dissected into the lower one third including the hair bulb (HB) and the upper hair shaft and sheaths (HS) including the bulge region. Microarray data was verified with qPCR and immunohistochemistry. Target effects were evaluated in vitro on human epidermal melanocytes (HEMs). In comparison to pigmented HS and HBs, several nucleotide excision repair (NER) family genes exhibited statistically significant lower expression both in non-pigmented HS and non-pigmented HB. These genes were identified as ERCC1, ERCC2, ERCC3, ERCC4, ERCC5, ERCC6, XPA, NTPBP, HCNP, DDB2 and POLH. Immunohistochemistry showed consistent results. By siRNA interference we also detected that a deficiency in ERCC3 in melanocytes reduced the ability to produce melanin in vitro. Our results suggest that loss of NER gene function may lead to DNA damage and mutation accumulation in melanocytes, which may possibly lead to cell death. Further, a loss of ERCC3 function may lead to reduced gene transcription and this in turn may lead to reduced melanin production ability. These results offer a new insight into the molecular changes that occur in non-pigmented HF and may also provide novel information with regard to melanogenesis and its regulation.
Project description:In order to facilitate understanding of pigment cell biology, we developed a method to concomitantly purify melanocytes, iridophores, and retinal pigmented epithelium from zebrafish, and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co-enrichment in melanocytes and retinal pigmented epithelium, as well as in melanocytes and iridophores. We found 214 genes co-enriched in melanocytes and retinal pigmented epithelium, indicating the shared functions of melanin-producing cells. We found 62 genes significantly co-enriched in melanocytes and iridophores, illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine-based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate-limiting substrate for purine synthesis, phosphoribosyl pyrophosphate, thus constituting a guanine cycle. The purification procedure and expression analysis described here, along with the accompanying transcriptome-wide expression data, provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types, and will be a useful resource for further studies of pigment cell biology.
Project description:In order to facilitate understanding of pigment cell biology, we developed a method to concomitantly purify melanocytes, iridophores, and retinal pigmented epithelium from zebrafish, and analyzed their transcriptomes. Comparing expression data from these cell types and whole embryos allowed us to reveal gene expression co-enrichment in melanocytes and retinal pigmented epithelium, as well as in melanocytes and iridophores. We found 214 genes co-enriched in melanocytes and retinal pigmented epithelium, indicating the shared functions of melanin-producing cells. We found 62 genes significantly co-enriched in melanocytes and iridophores, illustrative of their shared developmental origins from the neural crest. This is also the first analysis of the iridophore transcriptome. Gene expression analysis for iridophores revealed extensive enrichment of specific enzymes to coordinate production of their guanine-based reflective pigment. We speculate the coordinated upregulation of specific enzymes from several metabolic pathways recycles the rate-limiting substrate for purine synthesis, phosphoribosyl pyrophosphate, thus constituting a guanine cycle. The purification procedure and expression analysis described here, along with the accompanying transcriptome-wide expression data, provide the first mRNA sequencing data for multiple purified zebrafish pigment cell types, and will be a useful resource for further studies of pigment cell biology. mRNA profiles of zebrafish pigment cells were generated using Illumina GAIIX sequencing
Project description:Comparison between human adrenal cortex derived from adolescent female and human adrenal cortex derived from adolescent female affected by Primary Pigmented Nodular Adrenocortical Disease caused by germline mutation in PRKAR1a Keywords: other
Project description:Analysis of microarrays with transcriptome of human neural crest-originated stem cells (NCSC), highly metastatic BLM melanoma cell line and neonatal highly pigmented melanocytes (HPM) at whole genome scale demonstrated distinct differences among tested cell types.
Project description:Propose: We used next-generation RNA sequencing (RNA-seq) to characterize the transcriptional changes in primary human melanocytes during recessive Cole disease. Our patient carried missense mutation in the ENPP1 gene (c.358T>C; p.C120R). RNA-seq was performed using mRNA extracted from primary hypo- and hyper-pigmented melanocytes isolated from affected patient and melanocytes from his healthy heterozygous sibling and an aged- and ethnicity-matched control. Results: A pairwise fold-change comparison was performed and genes were computationally filtered using a cutoff of more than 2 fold change and P<0.01. We first compared hyper-pigmented melanocytes to each control individually and then overlapped the results to obtain a list of 1041 up-regulated and 692 down-regulated genes. The same analysis was done for hypo-pigmented melanocytes to found that 535 genes were up-regulated and 520 were down-regulated. Finally, to obtain a profile of the overall differential gene expression, down-regulated genes in hyper and hypo-pigmented cells were overlapped to identify 143 genes that were down-regulated in patient melanocytes compared to controls regardless of pigmentation status. Similar analysis was performed to obtain the list of 172 up-regulated genes. We selected 36 deregulated genes, most of which were associated with melanocyte development and pigmentation signaling pathways, and validated 32 of them by Q-PCR, indicating that our RNA-Seq data was accurate and reliable. Conclusion: Our study represents the first analysis of hypo- and hyper-pigmented primary melanocytes isolated from affected patient versus healthy controls in recessive Cole disese pathology.
Project description:Comparison between human adrenal cortex derived from adolescent female and human adrenal cortex derived from adolescent female affected by Primary Pigmented Nodular Adrenocortical Disease caused by germline mutation in PRKAR1a Keywords: other