Lecture 6 (G) PDF

Epigenetics Epigenetic: a process that affects gene expression without changing the underlying DNA sequence and inherited by daughter cells. Epigenetics means (on top of genetics) Epigenetic mechanisms control which genes are turned ON or OFF (expressed or not). It is a way for behaviors or traits t...

Epigenetics Epigenetic: a process that affects gene expression without changing the underlying DNA sequence and inherited by daughter cells. Epigenetics means (on top of genetics) Epigenetic mechanisms control which genes are turned ON or OFF (expressed or not). It is a way for behaviors or traits to pass down from one generation to the next without being encoded in the genetic code. Epigenetics mechanisms can be triggered by environmental factors Epigenetics DNA methylation The most common epigenetic mechanism is DNA methylation. DNA methylation is an epigenetic mechanism that occurs by the addition of a methyl (CH3) group to DNA and inhibits gene expression without changing the DNA sequence. Methylation occurs mainly over CG repeats (Cytosine followed by Guanine) bases in the genome. Epigenetics Normal DNA Gene expression Normal protein Thr Cys Thr Cys Genetics Ile Epigenetics DNA methylation mutation Mutant DNA Methylated DNA No gene expression Abnormal Gene expression Abnormal protein Thr STOP CH3 CH3 CH3 No protein Regullation of Gene Expression Control Gene expression Gene expression can be regulated at various steps RNA processing 2 RNA processing control 1 Transcriptional control DNA 3 mRNA transport and localization control Pre mRNA mRNA Degraded mRNA 4 mRNA degradation control 6 Protein degradation control Protein mRNA (A gene) 5 Translation control Nucleus Cytoplasm Inactive Protein 7 Protein activity control Active Protein Regullation of Gene Expression RNA processing control RNA processing is an important part of gene expression, as Coding region it is responsible for the generation of mature mRNA molecules that can be translated into proteins. One of the RNA processing mechanism is Alternative splicing. Alternative splicing is a cellular process in which exons 1 gene with from the same gene are joined in different combinations three exons leading to different mRNA molecules and, potentially, different protein products. ~ 95% of human genes undergo alternative splicing. Transcribed proteins 2 different transcripts Regullation of Gene Expression RNA processing control DNA Exon 1 5’ Regulatory element Intron 1 promoter Pre-mRNA Exon 2 Exon 3 Gene 2 3 mRNA (transcript 1) 1 2 mRNA (transcript 2) 1 3 mRNA (transcript 3) 1 2 4 mRNA (transcript 4) 1 2 3 3 4 4 1 gene 4 3’ Intron 3 Intron 2 1 Exon 4 4 Transcripts Regulation of Gene Expression Control Gene expression Gene expression can be regulated at various steps mRNA degradation/stability by MicroRNA 2 RNA processing control 1 Transcriptional control DNA 3 mRNA transport and localization control Pre mRNA mRNA Degraded mRNA 4 mRNA degradation control 6 Protein degradation control Protein mRNA (A gene) 5 Translation control Nucleus Cytoplasm Inactive Protein 7 Protein activity control Active Protein Regullation of Gene Expression MicroRNAs MicroRNAs (miRNAs) are small, non-coding RNA molecules that are involved in the regulation of gene expression. They are transcribed by RNA polymerases and then processed by a specific enzyme, which cuts them into small pieces of approximately 22 nucleotides in length. These pieces are then incorporated into a protein-RNA complex called an RNAinduced silencing complex (RISC), which binds to complementary messenger RNAs (mRNAs) and blocks their translation into proteins. MicroRNAs have been found to play a role in a wide range of biological processes, including development, differentiation, and disease. In humans, there are ~ 1000 miRNAs; most are expressed in certain cell types at a specific time point during development. Regullation of Gene Expression MicroRNAs miRNA DNA miRNA-Protein complex TRANSCRIPTION Target mRNA Pre-mRNA RNA PROCESSING mRNA miRNA miRNA X TRANSLATION Ribosome Polypeptide mRNA degraded Translation blocked Mutations (DNA variations) BIOL 111 Definition and Impacts on Phenotype A mutation or DNA variation is a heritable pathogenic alteration or change in the genetic material (DNA). It occurs through errors in DNA replication (mostly) or exposure to mutagenic agents. Changes in the DNA sequence associated with diseases are called pathogenic variants, while variants associated with normal phenotypic differences between individuals or do not affect the phenotype are called polymorphisms. Variations in the DNA sequences are the cause of the phenotypic differences between people (Lecture 4) DNA Variations Polymorphisms Pathogenic variants (Mutation) Marfan Syndrom Not associated with any phenotype Associated with different normal phenotypes Treacher Collins syndrome Associated with abnormal phenotypes (Diseases) Mutations (DNA variations) Types based on size Small or at DNA level Two types: - Substitutions: replacement of one nucleotide by another.. - Insertions and Deletions (Indels)* : insertion or deletion of one or more nucleotides. Molecular genetics Large or at chromosomal level Two types: - Numerical - Structural Cytogenetic *Indels can affect coding sequence of a gene, causing a frameshift mutation which shifts the way the sequence is read and can lead to protein expression changes. A frameshift occurs if the number of nucleotides is not a multiple if the number of nucleotides is not a multiple of three. Mutations (DNA variations) Normal BIOL 111 Structural Effects of DNA Variants on Protein DNA variants in coding regions can be divided based on the effect on the polypeptide sequence into two groups - Synonymous - Does not alter the amino acid - Nonsynonymous - Does alter the amino acid and can be subdivided into two types: - Missense: different amino acid - Nonsense: create a stop codon DNA Thr Synonymous Silent > Cys Cys Thr Protein Ile Nonsynonymous Missense Nonsense > Trp > Stop Mutations (DNA variations) BIOL 111 Normal Frameshift N OW Y O U C A N Thr H Ile Cys Thr S E E Insertion T N O W Y O U C TT A N S E * Thr Ser Thr Tyr * Frameshift Change in the reading frame Deletion Y N O W O U C A N S E E H * Thr Arg Val * Frameshift Leu Change in the reading frame E H Mutations (DNA variations) Variants nomenclature 1 Gene Symbol 2 3 DNA Seqnence change Protein change BRCA1 c.131G>A (p.Cys44Tyr) Coding seqeunce protein nucleotide change from G to A at nucleotide number 131 Amino acid change from Cysteine to Tyrosine at the amino acid number 44 Mutations (DNA variations) Types based on cell type (Somatic vs. Germline) Germline variants are changes in the genetic material that are passed down from parent to child. These variants are present in all the body's cells, including the germ cells, and can therefore be passed on to offspring. Somatic variants are changes that occur in a single body cell and cannot be inherited. These variants can only affect the tissues derived from the mutated cell. Mutations (DNA variations) Types based on cell type (Somatic vs. Germline) Germline mutations All cells carry mutation Transmitted to next generation Somatic mutations Not transmitted to next generation Only somatic cells carry mutation Functional Effects of Mutations on the Protein Functional effects of mutations on the protein Loss of function mutations (LoF) Gain of function (GoF) Functional Effects of Mutations on the Protein Loss of function mutations (LoF) Mutations cause either reduced activity or complete loss of gene product. For most genes, the cell can tolerate the loss of one allele and can function with the remaining 50% gene product from the normal allele (the exact quantity is not critical) Thus, diseases that are caused by loss of function mutations are inherited as recessive. (require both alleles to be mutated and make no product at all) Examples of loss of function mutations include mutations that cause cystic fibrosis, and sickle cell anemia. Functional Effects of Mutations on the Protein Phenotype Protein DNA Loss of function mutations (LoF) Normal Paternal allele Normal Maternal allele Normal Paternal allele Mutant * Maternal allele Normal Paternal allele Mutant ^ Maternal allele 50% normal protein 50% normal protein 50% normal protein 50% non-functional protein 50% normal protein 0% protein Normal Function NOT affected Normal Function NOT affected Normal Function NOT affected Normal Normal Normal * A mutation that leads to expression of protein with no or reduced activity ^ A mutation that leads to stop gene expression (no protein) Functional Effects of Mutations on the Protein Loss of function mutations (LoF) However, loss of function mutations can be dominant in case of Haplo-insufficiency mutations Dominant negative effect mutations Dominant negative effect mutations are mutations that cause a mutant protein to interfere with the activity of a wild-type protein. Functional Effects of Mutations on the Protein Loss of function mutations (LoF) Haplo-insufficiency mutations When the 50% gene product is not sufficient for the normal phenotype The phenotype is dosage sensitive and inherited as dominant. Functional Effects of Mutations on the Protein Loss of function mutations (LoF) Protein DNA Haplo-insufficiency mutations Phenotype Normal Paternal allele Normal Maternal allele Normal Paternal allele Mutant * Maternal allele Normal Paternal allele Mutant ^ Maternal allele 50% normal protein 50% normal protein 50% normal protein 50% non-functional protein 50% normal protein 0% protein Normal function NOT affected Normal function is affected Normal function is affected Normal Abnormal Abnormal * A mutation that leads to expression of protein with no or reduced activity ^ A mutation that leads to stop gene expression (no protein) Functional Effects of Mutations on the Protein Loss of function mutations (LoF) Dominant negative effect mutations Dominant negative effect mutations cause a mutant protein to interfere with the activity of a wild-type protein. The mutant protein interferes with the function of the normal protein produced by the normal allele The phenotype is inherited as dominant. Dominant negative effect is common in dimers or multimers, such as structural proteins like collagen. Mutations in collagen protein can lead to osteogenesis imperfecta. Functional Effects of Mutations on the Protein Loss of function mutations (LoF) Protein DNA Dominant negative effect mutations Phenotype Normal Paternal allele Normal Maternal allele Normal Paternal allele Mutant Maternal allele 50% normal protein 50% normal protein 50% normal protein 50% abnormal protein Normal function NOT affected Normal function is affected Normal Abnormal Functional Effects of Mutations on the Protein CLINICAL APPLICATIOM Loss of function mutations (LoF) Dominant negative effect mutations CLINICAL APPLICATION Osteogenesis Imperfecta (OI) - OI is a group of inherited diseases that lead to skeletal deformity and easy bone fractions. Most patients with OI have a heterozygous mutation in one of the Collagen genes. Healthy Bone Brittle Bone Functional Effects of Mutations on the Protein Gain of function mutations (GoF) Mutations cause either increased production of normal protein or modify the normal protein function/activity. In gain of function mutations, one mutant allele is enough to manifest the disease phenotype Two types of GoF mutations Mutations that increase the production of normal protein Mutations that change the normal protein function Most of gain of function mutations are inherited as dominant. Functional Effects of Mutations on the Protein Gain of function mutations (GoF) Phenotype Protein DNA Increase Production Normal Paternal allele Normal Maternal allele Normal Paternal allele Mutant Maternal allele 50% normal protein 50% normal protein 50% normal protein 100% normal protein Modify Function Normal Paternal allele Mutant Maternal allele 50% normal protein 50% protein with new function Normal function NOT affected Normal function is affected (more protein) Normal function is affected Normal Abnormal Abnormal Functional Effects of Mutations on the Protein Gain of function mutations (GoF) Change function Normal RAS Mutant RAS due to GoF mutation Regulated cell cycle pathway Upregulated cell cycle pathway CLINICAL APPLICATION RAS mutations in cancer Three ras genes work as cell cycle regulators by promoting the cell cycle when bound to GTP at the GTP form and inhibiting the cell cycle at the GDP form. Certain missense mutations lead to locking the protein in its GTP active form, which leads to constitutive activation of the cell cycle and cell proliferation (Oncogene activation) Controlled cell proliferation Increased cell proliferation Tumor Functional Effects of Mutations on the Protein Gain of function mutations (GoF) Increase Production CLINICAL APPLICATION Charcot-Marie-Tooth Disease (CMT) - Charcot-Marie-Tooth (CMT) Diseases are a group of hereditary neuropathies that are characterized by chronic motor and sensory neuropathy. - One type of CMT results from a duplication mutation of the PMP22 gene that leads to increased protein production. - The body can’t tolerate the increase in protein dosage cmtausa.org/understanding-cmt/what-is-cmt/