26 terms

PMBC5 - Regulation of Expression in Eukaryotes

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)
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
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