Lab
Cristofre Martin Lab
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Statement of Research Interest
Epigenetic Control of Gene Regulation:
The generation of a multicellular organism from a fertilized egg requires the establishment of multiple cell lineages with distinct properties. In vertebrates, the establishment of diverse cell lineages, each committed to a defined differentiation pathway, is believed in part to be dependent upon epigenetic processes that limit the array of genes that are expressed, and thus define the cellular phenotype.
DNA methylation occurs by the addition of a methyl-group to position 5 of cytosine by the enzyme DNA methyltransferase (MTase). This modification has several characteristics that make it an attractive mechanism that may account for much of the epigenetic control of gene regulation that occurs during normal embryonic development. DNA methylation affects the ability of transcriptional regulatory proteins to bind DNA, including repressor proteins like MeCP1 and MeCP2 (methyl-cytosine binding proteins), as well as affecting nucleosome positioning. Increases in DNA methylation are associated with gene silencing, and reductions in DNA methylation are often associated with gene activation. Furthermore, DNA methylation patterns can be preserved through the replication process by the action of maintenance methylases which use the replicated hemimethylated DNA as a template. DNA methylation can be removed by passive demethylation if methylation after replication is suppressed and possibly by active demethylation. As a result, DNA methylation is clonally heritable, stable, and reversible and, thus, satisfies the major requirements for an epigenetic mechanism that can control gene expression.
DNA methylation has been shown to be involved in a number of different biological processes (primarily in mammals) including genomic imprinting, X-chromosome inactivation, and gene silencing. Abnormal DNA methylation has been implicated in a wide range of human diseases including cancer.
We use molecular biology techniques such as PCR, mRNA differential display, in situ hybridization, and DNA chip microarrays to identify, clone and characterize genes that are expressed during zebrafish development and whose transcription is regulated by DNA methylation.
Epigenetic Toxins:
A number of compounds and heavy metals have been identified that can affect normal DNA chromatin structure by altering DNA methylation or histone acetylation. Many of these elements and compounds are found and used in our environment. Perterbations in these epigenetic processes such as DNA methylation and histone acetylation can lead to abnormal gene expression. We are using the zebrafish model system to evaluate the potential of these elements and compounds to affect gene expression in vivo and normal vertebrate development.
Transgenics:
Transcription of the zebrafish hsp10 and hsp60 (heat shock proteins) genes are driven by a single bi-directional promoter that is heat inducible. We are developing a transgene construct containing these promoter sequences. This construct will allow expression, in the zebrafish embryo, two genes simultaneous from a single transgene construct. Ectopic expression of the transgene can be achieved by heat shocking the zebrafish embryo, and expression of the transgene in a single cells or tissues can be achieved using lasers. We will use this transgene construct to address questions of gene function in the zebrafish embryo.
Related projects include promoter analysis, and investigating transgene applications for gene therapy.
The generation of a multicellular organism from a fertilized egg requires the establishment of multiple cell lineages with distinct properties. In vertebrates, the establishment of diverse cell lineages, each committed to a defined differentiation pathway, is believed in part to be dependent upon epigenetic processes that limit the array of genes that are expressed, and thus define the cellular phenotype.
DNA methylation occurs by the addition of a methyl-group to position 5 of cytosine by the enzyme DNA methyltransferase (MTase). This modification has several characteristics that make it an attractive mechanism that may account for much of the epigenetic control of gene regulation that occurs during normal embryonic development. DNA methylation affects the ability of transcriptional regulatory proteins to bind DNA, including repressor proteins like MeCP1 and MeCP2 (methyl-cytosine binding proteins), as well as affecting nucleosome positioning. Increases in DNA methylation are associated with gene silencing, and reductions in DNA methylation are often associated with gene activation. Furthermore, DNA methylation patterns can be preserved through the replication process by the action of maintenance methylases which use the replicated hemimethylated DNA as a template. DNA methylation can be removed by passive demethylation if methylation after replication is suppressed and possibly by active demethylation. As a result, DNA methylation is clonally heritable, stable, and reversible and, thus, satisfies the major requirements for an epigenetic mechanism that can control gene expression.
DNA methylation has been shown to be involved in a number of different biological processes (primarily in mammals) including genomic imprinting, X-chromosome inactivation, and gene silencing. Abnormal DNA methylation has been implicated in a wide range of human diseases including cancer.
We use molecular biology techniques such as PCR, mRNA differential display, in situ hybridization, and DNA chip microarrays to identify, clone and characterize genes that are expressed during zebrafish development and whose transcription is regulated by DNA methylation.
Epigenetic Toxins:
A number of compounds and heavy metals have been identified that can affect normal DNA chromatin structure by altering DNA methylation or histone acetylation. Many of these elements and compounds are found and used in our environment. Perterbations in these epigenetic processes such as DNA methylation and histone acetylation can lead to abnormal gene expression. We are using the zebrafish model system to evaluate the potential of these elements and compounds to affect gene expression in vivo and normal vertebrate development.
Transgenics:
Transcription of the zebrafish hsp10 and hsp60 (heat shock proteins) genes are driven by a single bi-directional promoter that is heat inducible. We are developing a transgene construct containing these promoter sequences. This construct will allow expression, in the zebrafish embryo, two genes simultaneous from a single transgene construct. Ectopic expression of the transgene can be achieved by heat shocking the zebrafish embryo, and expression of the transgene in a single cells or tissues can be achieved using lasers. We will use this transgene construct to address questions of gene function in the zebrafish embryo.
Related projects include promoter analysis, and investigating transgene applications for gene therapy.
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