Lecture theme 3 Role of chromatin structure in gene control Flashcards
Experimental proof that chromatin structure plays a role in gene-specific expression : DNase1 sensitivity assay.
You need an X-specific probe to do the experiment. isolate CHROMATIN from both cell types … treat both with a range of DNase1 concentrations, high to low
DNase1 cuts preferentially in the linker region of the nucleosome
chains …. accessible when DNA is in beads-on-a-string structure
if gene X is packed densely in kidney cells (will not be transcribed), the gene will not be digested at low [DNase1]
If the X-gene is loosely packaged in liver cells (ready or committed for transcription), gene will be digested at low [DNase1]
1) How is methylation experimentally detected?
2) Other assays suitable for genome-wide analysis
1
How is methylation experimentally detected? Principle of assay: certain RE can distinguish between methylated and unmethylated sites on DNA.
site CCGG is recognised by both MspI and HpaII
MspI digests unmethylated or methylated DNA
HpaII only digests unmethylated DNA
2
Other assays suitable for genome-wide analysis
•DNA is treated with sodium bisulfite.
•sodium bisulfite has no effect on methylated C’s, but converts all unmethylated C’s to U’s
•C’s that are not converted to uracil are therefore methylated.
DNA is methylated by methyl-transferase enzymes.
• Dnmt3a or b for de novo (new) methylation of unmethylated sites
• Dnmt1 maintains the methylation pattern following DNA replication.
-recognises and methylates half- methylated sites
-allows methylation patterns to be propagated stably throughout cell divisions
Slide 18
How to investigate if chromatin structure plays a role in gene regulation?
Chromatin of genes actively transcribed or committed to transcription has less dense DNA conformation than genes not committed for transcription
Less dense DNA provides access to enzymes such as DNase1
Compare chromatin structure in active vs non-active by analysing the sensitivity of chromatin to DNase1 digestion.
