The regulatory Genome - Week 3

Question 1

regulatory genome

Answer 1

non-coding genome

Question 2

in humans the non-coding genome represents

Answer 2

99% of the genome

Question 3

only 1% of the genome is

Answer 3

coding

Question 4

Evidence for function in the non-coding genome

Answer 4

biochemical evidence, genetic evidence, evolutionary evidence,

Question 5

Evidence for function in the non-coding genome: biochemical evidence

Answer 5

Genome wide studies i.e. ENCODE - identifying molecular activity (transcription, transcription factor binding), up to 80% of the genome functional

Question 6

Evidence for function in the non-coding genome: genetic evidence

Answer 6

Function defined by - phenotypic consequence of mutation, up to 15% of the genome ‘functional’

Question 7

Evidence for function in the non-coding genome: evolutionary evidence

Answer 7

conservation of sequence across evolution, up to 10% of the genome ‘functional’

Question 8

What does the non-coding genome contain?

Answer 8

Promoters, enhancers, silencers, insulators, noncoding RNA agents (microRNAs, piRNAs, structural RNAs, regulatory RNAs)

Question 9

Non-coding functional elements associated with

Answer 9

distinctive chromatin structures that display signature patterns of histone modifications, DNA methylation, DNase accessibility and transcription factor occupancy.

Question 10

Enhancers - how are they defined

Answer 10

A set of clustered short sequence elements that stimulate the transcription of a gene and whose function is not critically dependent on their precise position or orientation

Question 11

Enhancers - mechanism of action

Answer 11

serve as sites for pre-initiation complex (PIC) formation (complex of dozens of proteins including RNA polymerase and general transcription factors necessary for transcription.)

By looping of the intervening DNA to make contact with the target gene promoter the PIC is delivered to the required site and transcription proceeds.

This loop formation might increase the concentration of PIC at the target gene and thus transcription. This is one model whereas there is also evidence for other models:
• Looping results in relocation of target gene within nucleus to a ‘neighbourhood’ within the nucleus more favourable for transcription (so called transcription factories).
• Looping results in recruitment of transcription elongation factors to promoters that can increase rates of transcription elongation (the step after transcription initiation)

Question 12

Enhancers - what evidence is there that they are important to human health?

Answer 12

• Genetic evidence can be provided by finding mutations causing disease. For Mendelian disease mutations in a limb enhancer for SHH is a prototypical enhancer.
• Single nucleotide polymorphisms associated with risk of developing complex, polygenic disease have also been shown to be enriched in disease-relevant cell type enhancers (e.g. type 2 diabetes and pancreatic islet enhancers)
• Evolutionary conservation of enhancer sequences is also good evidence of importance to human health
• As is evidence of paucity of rare variation across human populations in human accelerated regions (regions with evidence of accelerate divergence from other primates and thus likely important for human-specific traits) Most HARs are believed to be enhancers.

Question 13

Enhancers - What methods are used to identify them?

Answer 13

• Chromatin Conformation Capture (to capture looping interactions)
• Reporter Gene assays (e.g. luciferase) to show ability of sequence to increase expression of reporter gene transfected into cells
• ChIP-Seq (identify DNA binding sites for transcription factors and DNA associated with histone modifications associated with enhancer function (e.g. H3K4me1)
• DNase-Seq (treat DNA with DNase I that cleaves DNA in open chromatin (e.g. enhancer) and sequence these fragments)

Question 14

Enhancers - What are the current and future directions for research? (ie what remains to be found out and why is this important?)

Answer 14

• Cell sorting to identify spatially and temporally restricted enhancers (e.g. Human Cell Atlas)
• Is there an enhancer code? (the sequences of enhancers does not give good discrimination but could other parameters (ChIP-Seq binding) provide better discrimination?
• To what extent can human disease (Mendelian and complex, polygenic) be explained by genetic variation in enhancers?

Question 15

Insulators - how are they defined?

Answer 15

DNA element that acts as a barrier to the spread of chromatin changes or the influence of cis¬-acting elements

Question 16

Insulators - What is/are there mechanism of action?

Answer 16

• Insulator sequences have been identified by their ability to block the activity of enhancers to increase gene expression when placed between promoter and enhancer. This may be by preventing the spreading of heterochromatin or the looping and contact between enhancer and promoter.
• Insulators have been shown, in vertebrates, to be enriched in CCCTC sequence and bind CCCTC-binding factor (CTCF). CTCF plays a key role in establishing topologically associating domains (TADs). Interactions within TADs are enabled whilst between TAD interactions are prevented

Question 17

Insulators - What evidence is there that they are important to human health?

Answer 17

• CTCF-binding sites that define insulator sites are frequently mutated in cancer
• Microdeletions in the an insulator sequence that regulates IGF2 and H19 imprinting results Beckwith-Wiedemann syndrome

Question 18

Insulators - What methods are used to identify them?

Answer 18

• Reporter genes inserted either flanked by insulator sequences or not. If flanked sequences are insulating then the reporter gene will be active whether inserted into heterochromatin or euchromatin.
• The ability of an insulator sequence to block promoter: enhancer interactions can be assayed by placing the tested sequence in between promoter and enhancer.

Question 19

Insulators - What are the current and future directions for research? (ie what remains to be found out and why is this important?)

Answer 19

• In species such as Drosophila many more proteins that bind insulators are known than for humans. Are there more insulator proteins in humans and will this explain the lack of CTCF-binding sites at some topologically associating domains (TADs)?

Question 20

Non-coding RNAs - How are they defined?

Answer 20

Mature RNA transcript that is not translated to make a polypeptide

Question 21

Non-coding RNAs - What is/are there mechanism of action? List the types of non-coding RNA

Answer 21

MicroRNAs (miRNAs), Transfer RNAs (tRNAs), Ribosomal RNAs (rRNAs), small nuclear RNAs (snRNAs), long non-coding RNAs (lncRNAs), Circular RNAs (circRNAs), Piwi-interacting RNAs (piRNAs)

Question 22

Non-coding RNAs - What is/are there mechanism of action? MicroRNAs (miRNAs)

Answer 22

small (~20nt) single-stranded RNAs that bind, via complementary base pairing, to transcripts and cause translational repression and/or mRNA degradation

Question 23

Non-coding RNAs - What is/are there mechanism of action? Transfer RNAs (tRNAs)

Answer 23

adaptor RNAs with a classic cloverleaf structure that carry amino acids covalently linked to their 3’end and also containing an anticodon sequence that base pair with the codons on mRNAs. Hence crucial for translation.

Question 24

Non-coding RNAs - What is/are there mechanism of action? Ribosomal RNAs (rRNAs)

Answer 24

Ribosomes are protein:RNA macromolecular complexes that catalyse the reaction of joining amino acids together. rRNA makes up ~80% of RNA (by mass) in a human cell.

Question 25

Non-coding RNAs - What is/are there mechanism of action? o Small nuclear RNAs (snRNAs)

Answer 25

~150nt long RNAs that most famously bind to regions near splice sites and make up part of the spliceosome, the complex that removes introns and joins exons together in the process called splicing.

Question 26

Non-coding RNAs - What is/are there mechanism of action? Long non-coding RNAs

Answer 26

long RNAs (>200nt) can play crucial roles in regulating gene expression by recruiting chromatin-modifying complexes to modify histones and/or DNA methylation.

Question 27

Non-coding RNAs - What is/are there mechanism of action? Circular RNAs (circRNAs)

Answer 27

back splicing’ results in formation of circular RNAs that may sequester miRNAs and thus regulate gene expression.

Question 28

Non-coding RNAs - What is/are there mechanism of action? Piwi-interacting RNAs (piRNAs)

Answer 28

active in germ cells to repress transposon (mobile DNA) activity and crucial for germ cell and stem cell development.

Question 29

Non-coding RNAs - What evidence is there that they are important to human health?

Answer 29

• Their involvement in ubiquitous cellular processes (e.g. protein synthesis (tRNAs & rRNAs), splicing (snRNAs) and gene regulation (miRNAs, lncRNAs)). Their abundance and numbers also argue for importance

Question 30

Non-coding RNAs - What methods are used to identify them?

Answer 30

• Size or characteristic selection of RNA, conversion to complementary DNA and NGS.

Question 31

Non-coding RNAs - What are the current and future directions for research? (ie what remains to be found out and why is this important?)

Answer 31

• Projects such as the Human Cell Atlas will reveal whether low-level expression of ncRNAs in groups of cells/tissues is due to a small number of cells expressing high levels or generally low level expression across all cells.
• Cell-specific expression and involvement in disease pathology offer tremendous potential for using ncRNAs as biomarkers and to be targeted by therapeutics (see table below).

Question 32

Promoters - how are they defined?

Answer 32

• A combination of short sequence elements, usually just upstream of a gene, to which RNA polymerase binds so as to initiate transcription of the gene

Question 33

Promoters - what is there mechanism of action?

Answer 33

• General transcription factors sequentially assemble at the core promoter to recruit and activate RNA polymerase to unwind DNA and begin transcription.
• General transcription factors may bind to TATA box or BRE elements shown below. But it is important to note that not all promoters contain these sequence motifs (i.e. there presence and spacing is sufficient but not necessary).

Question 34

Promoters - what evidence is there that they are important to human health?

Answer 34

• Mutations in promoter sequences of many genes are known to cause a plethora of Mendelian diseases
• Indicative of importance binding of transcription factors, the binding sites and the characteristic epigenetic marks are highly conserved from mouse to human

Question 35

Promoters - What methods are used to identify them?

Answer 35

• A reporter gene assay with the candidate promoter region upstream of the reporter gene is truncated and sometimes split to identify smaller regions that can recapitulate most or all the transcriptional activity
• More high-throughput genome-wide techniques to identify transcriptional start sites and core promoters are now in use, some of which make use of the chemical structure of the 5’cap present at the start of mRNAs

Question 36

Promoters - What are the current and future directions for research? (ie what remains to be found out and why is this important?)

Answer 36

• To predict the phenotypic consequences of changes in transcription factor activity it is necessary to understand how certain transcription factors are important to promoter but not enhancer function. Even greater throughput assays are required to understand the transcription factors and binding sites necessary in different cell types.

Question 37

Silencers - How are they defined?

Answer 37

• Combination of short DNA sequence elements that suppress the transcription of a gene

Question 38

Silencers - what is there mechanims of action?

Answer 38

• Silencers recruit negative transcription factors (so called repressors) and co-repressors including Polycomb repressive complex. In another cell type the same sequence may actually act as an enhancer

Question 39

Silencers - what evidence is there that they are important to human health?

Answer 39

• Lack of genome-wide approaches to identifying silencers means there are only isolated examples of their importance (e.g. Deletion of D4Z4 repeats affecting expression of genes at chromosome 4q35 causing Fascioscapulohumeral Muscular Dystrophy (FSHD)).
• The dual activity model for sequences that can act as silencers in one cell type and enhancers in another mean that much of the evidence for importance of enhancers could also apply to silencers (e.g. evolutionary conservation, mutations causing disease) but this remains to be determined.

Question 40

Silencers - What method is used to identify them?

Answer 40

• Historically, reporter gene assays have been used to identify silencers. In these a sequence with putative silencer activity is inserted upstream/downstream (like enhancers they are orientation-independent) of a reporter gene being driven by a relatively strong proximal promoter
• Very recently, a more high-throughput method has been published which involves transfecting cells, stimulated to apoptose, with putative silencer sequences and identifying cells that survive (due to the silencing of the pro-apoptotic Caspase-9 gene)

Question 41

Silencers - What are the current and future directions for research? (ie what remains to be found out and why is this important?)

Answer 41

• As eluded to genome-wide identification of silencers spatially and temporally will increase our understanding as to how important they are and their overlap with enhancers.
• This in turn will enable the phenotypic effect of genetic variation (somatic and germline) in these elements to be better understood.