W12L1

Question 1

What are the different regions of the genome or DNA?

Answer 1

Genes – introns and exons

Enhancers
- Tell the gene “when”, “where”, and “how much”

Promoters
- Tell the gene “when”, “where”, and “how much”

Intergenic regions- Garbage? Regulatory regions? Transcribed areas?

Question 2

Promoters

Answer 2

Position is fixed – upstream (5’) of the Transcriptional Start Site (TSS)

Generally 150-500 bp in length

contains the consensus DNA sequences TATA/CAAT

Proximal control elements such as TFIIB recognition element (BRE) and initiator sequences (INR)
- INR is where polymerization will bind to and start transcription (20 base pairs up from where transcription DNA is)

Binds General (Basal) Transcription Factors

May bind tissue specific transcription factors (TSTF)

Question 3

TFII proteins

Answer 3

TFIID binds the core promoter region (binds to TATA box) and may bind additional sequences near the TSS, such as the INR (initiator sequence)

TFIIA and TFIIB help to stabilize TFIID binding
- TFIIB helps recruit RNA polymerase II

TFIIF binds RNA PolII and TFIIB which aids in recruitment of RNA polymerase II (PROMOTER CAN REMAIN IN THIS STATE…WHY?)

TFIIE and TFIIH are recruited. These factors help to melt and unwind the promoter region, and activate the polymerase to begin RNA synthesis
- TFIIH phosphorylates RNA polymerase II, the polymerase releases from the general transcription factors leading to transcriptional elongation

Tissue specific transcription factors often aid in long range interactions between different chromosomal regions

Question 4

Enhancers

Answer 4

Working on same chromosome = Cis regulation

Working across chromosomes = Trans regulation

Enhancers
- Can be upstream or downstream
- Can be on different chromosomes (trans-regulation)
- “position” and direction independent (within reason)
- Affect DNA folding and interactions

Question 5

Nucleosomes

Answer 5

DNA is wrapped around histone protein complexes that allows for more efficient packaging, regulation of gene expression and interaction between distant regions of DNA

• heterochromatin is tightly bound, euchromatin is loosely bound
• enhancers help switch between heterochromatin and euchromatin

Binding to histones to form nucleosomes and also binding to non-histone scaffolding proteins to organize into larger looping structures

Question 6

Chromatin

Answer 6

Have identified that chromatin is non-randomly distributed within the nucleus in interphase
- Distribution is cell type specific

Identified gene sequences (LADs) that bind to lamin associated proteins —> can repress gene expression

Why would the nucleus of cells spend so much time organizing chromatin into regions?

Question 7

DNA Looping

Answer 7

DNA looping can bring enhancer and promoter regions to regulate gene expression
- Binding of co-activators or co-repressors to the same region will regulate expression from a distance
- GRIN2B expression can undo the DNA looping

DNA looping and long range interactions is a mechanism for coordinated gene expression
- topologically associated domains (TAD, which are regions of DNA brought close together) separated by architectural proteins

Question 8

Haemaglobin expression

Answer 8

Co-linear expression of globins

Two chromosomes with genes that must be transcribed in parallel
- want heterodimer (i.e. between alpha and beta)

How does the erythrocyte accomplish this task?

RNA FISH (Fluorescent in situ hybridization)
- Identifies the transcription of two haemoglobin genes
- Localized to the same area within the nucleus

Question 9

Promoter analysis (aka Promoter Bashing): Reporter genes

Answer 9

Reporter genes

• Easily detectable
• Not normally expressed in the cells or tissue to be analyzed
• Not quickly degraded
• Not subject to post-transcriptional or post-translation regulation
• Examples include green fluorescent protein (GFP) or other fluorescent proteins (YFP, mCherry, etc.), luciferase, and β-galactosidase

Question 10

Promoter-reporter assays - Luciferase

Answer 10

Emission of light

Stable

Does not affect the cells

QUANTIFIABLE

Question 11

Promoter-reporter assays - LacZ

Answer 11

Very stable

Easily detectable

In vivo analysis

Not for quantification

Answers:
Does this promoter region recapitulate COMPLETE expression in the pancreas? or PARTIAL

Question 12

Promoter analysis - Why is this important?

Answer 12

May determine the factors that promote expression

May aid in defining genetic mutations that have a distinctive phenotype but are not within a gene

Can be used as a tool to drive the expression of other genes in a cell, tissue and time specific fashion

E.g. Viral introduction for gene therapy