Expressing the Genome Flashcards
Describe the anatomy of a gene
Towards the 5’ end there are upstream enhancers. Then there are promoters closer to the gene, then a TATA box which is upstream of the 5’ UTR. Following the 5’ UTR there is the coding region which is made up of exons and introns. Downstream of the last exon is the 3’ UTR.
Describe what is meant by gene expression and outline the process
Gene expression is the process by which genetic information is used to create a functional gene product ie. protein/RNA.
In initiation, basal transcription machinery assembles at the promoter which usually have a TSS, TATA box and regulator sequences like enhancers, silencers. Core promoter is TSS and TATA. Basal machinery is RNA PII and TFIIB,D,E,F,H. TFIID has TATA binding protein and 11 TBP associated factors and is the first to bind to promoter. The rest of the TFs and RNA PII assemble forming the initiation ocmplex factors. Translation begins (elongation) and processing is co-transcriptional. 5’ capping (adding 7-methylguanine) starts as soon as trancription does, splicing and editing also all occur. Polyadenylation is an inherent part of the termination process.
How can we measure gene expression?
qPCR quantifies a specific transcript which we have created cDNA primers for which allows it to be amplified.
Gene expression microarrays use fluorescently labelled RNAs to hybridise to transcript specific oligos on a solid support which allow detection and quantification of transcripts in an mRNA sample.
RNA-seq is a next gen technique where a cDNA library is created (from the mRNA) and sequenced. Tells us which genes are expressed and the read number tells us the expression level.
How does DNA methylation change gene expression?
Occurs at carbon-5 of cytosine, forming 5-methyl cytosine (5th base - so common 1.5% of genome). This disrupts the DNA double helix, inhibiting transcription (forming heterochromatin). CpG dinuclotides methylated in islands at promoters is associated with gene silencing. Methylation occurs by DNA methyltransferases.
What is genomic imprinting?
Genomic imprinting is when one copy of a gene is silenced by epigenetics, so only one allele is expressed. Combined with mutations, this can lead to disease because the second allele won’t compensate for the mutated one.
What is Prader-Willi Syndrome?
SNRPN gene on chromosome 15 has its paternal allele deleted and the maternal is silenced by imprinting, this leads to a lack of SNRPN protein which causes hyperphagia, and motor skills are developmentally delayed due to a lack of muscle tone.
What is Angelman syndrome?
UBE3A gene on chromosome 15 has a deletion of maternal allele and paternal is silenced. Causes nervous system disorders characterised by developmental disabilities, seizures, speech deficits, and motor oddities.
How do histone modifications influence the chromatin status of DNA? Give some examples of histone modifications.
Post-translational histone mods can turn DNA into heterochromatin (tightly coiled) or euchromatin (loose).
Lys-27 acetylation promotes transcription.
Lys-27 trimethylation suppresses transcription leading to large ares of inactive chromatin.
Lys-4 trimethylation of H3 activates promoter.
Lys-9 di/trimethylation silences promoter.
How is chromatin organised in the nucleus?
Chromatin looping allows contact over large distances (tissue specific enhancers are conserves regions usually hundreds of Kbs away from core promoter). The active chromatin is compartmentalised near the nucleus, and the inactive near the periphery. Topologically Associated Domains are self-interacting regions of the genome, where most enhancer-promoter pairs occur. Genes within the TAD are brought together in a cell specific manner, influencing expression. The chromosomes also segregate into distinct territories.
Explain the process of ChIP-seq
Chromatin Immunoprecipitation with high-throughput sequencing identifies where proteins of interest (TFs, histone tail mods, etc.) bind DNA. The proteins bind to DNA, and chemical cross links are formed between the protein and the DNA. The DNA is fragmented and the protein and fragment are isolated by antibodies. The crosslinks are removed, releasing the protein, and the DNA fragment is sequenced.
What is Chromosome Conformation Capture?
3C identified regions of physical contact in the genome. Chromatin strands are treated with formaldehyde which cross links them at points of contact. They are then digested with restriction endonucleases. The free ends are then ligated together and the cross link is removed. The circular strand of DNA is then sequenced.
What is the hierarchical organisational structure of chromatin and what can disruption of this structure do?
Hetero/euchromatin –> A/B compartments –> TADs and chromatin loops. If there are mutations in proteins which uphold this structure, there could be pathogenic alterations.
What happens when chromatin loops are compromised?
The loss of an enhancer-promoter pair could result in the loss of enhancer function and alter the binding of TFs to the gene, resulting in contribution to disease progression. Occurs in T cell ALL, asthma, heart diseases
What happens when stable TADs are disrupted?
TAD boundary deletions can cause rewiring of promoter-enhancer interactions, which means that enhancers from other domains to activate other genes, leading to aberrant gene expression. Occurs in F-syndrome and sex reversal.
What happens when chromosome territories are disrupted?
The emergence and dissolution of chromosomal territories is seen in several cancers. When a translocation brings the coding sequence of one gene into the regulatory environment of another genomic region, the transcription of that gene can be abberantly activated/silenced.
