PMBC5 - Regulation of Expression in Eukaryotes

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What are the 6 main mechanisms involved in regulation of gene expression in eukaryotes

transcriptional control, mRNA processing, mRNA transport, mRNA stability, Ribosomal selection, protein stability and processing

What are the 4 different mechanisms for control of differential gene expression unique to eukaryotes?

Cis-regulatory sequences, transcription factors (trans-acting), epigenetics regulation (changes in chromatin conformation by gene methylation and histone deacetylation), and small nuclear RNAs

1. Describe the sequential events and the proteins/enzymes involved in producing epigenetic gene repression

cytosine methylated to 5-methyl-cytosine by DNA methyl transferase - methyl group becomes flag for methyl-cytosine binding protein (MeCP2). Dnmt3 binds to methylated DNA and recruits MeCP2 and histone deacetylase (HDAC) protein to bind to methylated DNA and represses transcription by deacetylation the chromatin affecting its integrity. When DNA is replicated it becomes hemi-acetylated DNA, recruits Dnmt1 which methylates the new strand of DNA and recruits HDAC again and transcription is repressed again.

What are some modifications of histones

acetylation (HATs - transcriptionally active), deacetylation (HDACs - transcriptionally inactive), methylation, ubiquitination, phosphorylation

Explain globin-switching

during the first 6 weeks of fetal development, the promoter on the gamma globin gene is methylated which causes inactive gamma globin gene. At 12 weeks there is a switch, where the epsilon globin gene becomes methylated and inactive and the gamma globin becomes demethylated and active. Creates difference in fetal hemoglobin subunits

2. Immunodeficiency-centromeric instability-facial anomalies (ICF) syndrome

mutation in Dnmt3b gene

Rett syndrome

mostly females (on X chromosome, only affects Kleinfelter-XXY boys, otherwise causes death) - mutation in the X-linked gene methyl-cytosine binding protein 2 (MeCP2)

Genomic imprinting

monoallelic expression - one of the parental alleles is silenced and not expressed (affects several hundred genes). If father is mutant of maternally imprinted gene, all children will be mutants, if father is mutant of paternally imprinted genes, all children are normal. Imprinting (methylation that shuts off gene expression) occurs in gametogenesis

3. Prader-Willi syndrome

Paternal deletion -> no snRNP gene product

Angelman syndrome

Maternal deletion product -> no ubiquitin gene product

Explain how cancers can be caused by faulty gene imprinting

abnormal DNA methylation - IGF2 (insulin like growth factor) and IGF2 receptor have shown to be abnormally methylated and show abnormal gene expression. Genes become biallelic and begin producing too much of their gene products. Treat with drugs that interfere with DNA methylation - 5-aza-cytidine, and histone deacetylation (sodium butyrate)

5-aza-cytidine

affects DNA methylation - anti cancer

Sodium butyrate

affects histone deacetylation - anti cancer

What are the environmental factors that have been shown to influence or disrupt epigenetic gene silencing

Temperature (DNA methylation in zebra fish transgenes), toxins (arsenic and nickel cause hypomethylation), maternal care (modification of glucocorticoids receptor methylation), diet (folate and homocysteine - methyl donators), hypoxia (demethylation of HRE in erythropoietin gene required for activation)

Yellow agouti mice

mother's prenatal diet lacked methyl donors - folate - agouti gene becomes unmethylated and active - turns yellow. Normal mice's mother had folate supplements - agouti gene is methylated and inactive.

4. Define cis-regulatory elements and trans-acting factors

cis - sequences on the gene where proteins bind e.g. promoter/operator, enhancers/silencers. Trans - nuclear proteins that bind to promoter or enhancer sequences and stimulate transcription

5. Explain the significance of genes sharing cis-regulatory elements of similar sequence

ability to turn on a number of genes at the same time - stage specific expression (embryonic development), tissue specific expression, and response to external stimuli (hypoxia/hormonal/heat response elements)

6. Explain the important functional domains of typical trans-acting, DNA binding proteins

activation/repression domain (for transcriptional activation via interaction with basal transcription machinery), dimerization domain, DNA binding domain

7. Explain how activator and repressor proteins interact with each other to regulate gene transcription

folding of DNA can result in interactions between enhancer or repressor proteins bound to distant cis elements and the basal transcription machinery

Competition for enhancer sequence

binding of repressor to enhancer sequence blocks binding of activator - reduce transcription levels

Quenching

activator binds repressor to DNA binding domain or activation domain - prevents transcription

Explain the regulation of iron storage

High iron causes Iron response element binding protein (IRE-BP) to be inactive and ferritin is translated by IREs on 5' end and transferrin receptor (TfR) mRNA is degraded by IREs on 3' end. Low iron, IRE-BPs are active and bind to IREs at 5' end of ferritin to prevent translation initiation, bind to IREs on 3' end of TfR to protect the mRNA from being degraded.

8. Explain the various steps involved in the processing of miRNA

transcription of microRNA genes with internal complimentary --> microRNA cluster --> gets processed by Drosha enzyme to form long double stranded stem loops --> dicer associates with dsRNA and chops into small miRNA (20-25 nucleotides) which forms single stranded RNA by associating with RISC proteins

9. Describe how miRNA regulates gene expression

by binding to sequence elements in the 3'UTR of a specific mRNA and preventing interaction of translational machinery with the 7-methyl-guanosine cap structure (preventing mRNA from getting translated)

How are miRNA related to cancer

miRNAs act on tumor suppressor genes - too many miRNAs, always inhibiting tumor suppressor genes

10. Discuss the potential role of RNA interference in gene therapy

siRNA inhibits BCl2 (inhibitor of apoptosis) - kills cancer cells

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