CSB349H1 Lecture Notes - Lecture 6: Small Interfering Rna, Cpg Oligodeoxynucleotide, Myc
14 May 2018
School
Department
Course
Professor
Lecture 6(c): DNA Methylation
Recruiting DNMT3 Proteins – De Novo Methylation:
A. Recognition Motifs:
o DNA methyltransferases may have recognition motifs
that allow them to recognize DNA and allow them to
bind and methylate that region
B. Transcription Factors:
o Transcription factors bind to promoters and specific
DNA sequences – may also be capable of binding to
DNA methyltransferases to specific DNA regions
C. Gene Silencing Pathways:
o Transcriptional gene silencing pathways may be involved in targeting DNA
methyltransferases via small interfering RNA (RNAi) to specific DNA regions to
undergo methylation
Myc Recruiting Dnmt3a:
• Myc is a transcription factor that functions to repress
p21Cip1
transcription through the
recruitment of Dnmt3a
Experiment 1 – Does the Myc and Dnmt3a physically interact?
Pull Down Assay:
• Gene of interest is expressed in E.Coli cells to allow
the artificial production of proteins
o Protein of interest will be purified via a tag –
will recognize a tag found on the protein
§ Glutathione-S-Transferase (GST) is a tag used to purify Dnmt3a
ð If Dnmt3a is interacting with another protein (i.e. Myc transcription factor) it will also be pulled
down via the tag; known as co-purification
o Myc binds to the PHD domain of Dnmt3a in pull down assays
Experiment 2 – Does Myc and Dnmt3a interact at the p21Cip1 promotor?
ChIP Experiment:
• DNA is crosslinked and antibody stained to recognize proteins (Myc/Dnmt3a)
o Fragments are Immunoprecipitate and specific DNA
fragments (promotor) are identified via PCR
ð In the presence of Myc, both Dnmt3a and Myc are found on the promotor of p21Cip1
o If Myc is absent then Dnmt3a is not found on the promoter
Experiment 3 – Does recruitment of Dnmt3a lead to changes in methylation?
Bisulfite Sequencing:
• Looking at 5 CpG sequences along the promoter:
o CpG sequences are found methylated in the presence of Myc
o CpG sequences are found unmethylated in the absence of Myc
Document Summary
Recruiting dnmt3 proteins de novo methylation: recognition motifs: Lecture 6(c): dna methylation: dna methyltransferases may have recognition motifs that allow them to recognize dna and allow them to bind and methylate that region, transcription factors, transcription factors bind to promoters and specific. Dna sequences may also be capable of binding to. Dna methyltransferases to specific dna regions: gene silencing pathways, transcriptional gene silencing pathways may be involved in targeting dna methyltransferases via small interfering rna (rnai) to specific dna regions to undergo methylation. Myc recruiting dnmt3a: myc is a transcription factor that functions to repress p21cip1 transcription through the recruitment of dnmt3a. Pull down assay: gene of interest is expressed in e. coli cells to allow the artificial production of proteins, protein of interest will be purified via a tag will recognize a tag found on the protein. Glutathione-s-transferase (gst) is a tag used to purify dnmt3a.
