Download Horak - Blumberg Lab

Tori Tucker and Minh Nguyen
D145 Presentation
February 16, 2017
Cell Cycle -- G1/ S Phase focus
Paper Main Ideas
- Yeast: Saccharomyces cerevisiae transcription factor SBF (Swi4-Swi6 cell
cycle box binding factor)
- SBF = major transcription factor = regulates start of G1/S phase of cell cycle
- SBF binding targets: HCM1, PLM2, POG1, TOS4, TOS8, TYE7, YAP5,
YHP1, YOX1 = promoters of other transcription factors
GOAL: To demonstrate that these factors are targets of SBF by chIp-chip and to
also determine targets of transcription factors that interact with SBF
SBF = Protein
Swi4= a subunit part of SBF4 → binds to target promoters
chIp-chIp Method Overview
- Determine protein-DNA interaction...What do you need?
- Protein of Interest= transcription factors → SBF (Swi4 subunit) → HA-Swi4
- DNA= transcription promoters → GAT2, HCM1, NDD1, PDR1, PLM2, POG1, SOK2, TOS4,
TOS8, TYE7, YAP5, YHP1, YOX1
- Antibody = Anti HA (immunoprecipitate HA-Swi4)
- PCR Primers= Needed to amplify immunoprecipitated DNA that interacts with HA-Swi4
- Combines protein immunoprecipitation (IP) and DNA Microarray (chip)
GOAL: to locate protein binding sites that can help identify functional elements in
the genome.
Generating a HA Tagged
Protein
Plasmid: Contains Swi4 and HA tag
→ transcribe together
- How to get in cells?
- Overexpress plasmid by transfection
- HA-Swi4 incorporate into yeast
genome → transcribed → bind to parts
of DNA
Source:
http://blog.addgene.org/plasmids-101mammalian-vectors
Swi4
HA
Step 1: Allow protein
(Swi4-HA) to bind to
DNA promoters
Step 5: Can
use a
microarray
to look at
expression
or a gel (ie.
Southern
blot)
Step 2: DNA
Step 3: Use antibody
Fragmented as DNA
to bind to HA on Swi4
or DNA-Protein
Step 4:
Protein
removed,
DNA
amplified
(PCR)
Swi4-HA Binding
Results
- PCR → Product amplified → gel
electrophoresis
- Lane 1: untagged DNA → DNA that
did not interact with Swi4
- Lane 2: DNA interacted with HASwi4
- Lane 3: Total DNA
- Left side: designates the the band
we are looking at (ie. pCLN2)
- Fold Enrichment: based on how
much the promoter interacted with
Swi4
Determining targets of transcription factors that
interact with HA-Swi4
- Transcription Factors: HCM1, TOS4, YHP1
- Swi4 interacts with HCM1, TOS4, YHP1 → What parts of yeast DNA do
HCM1 TOS4 and YHP1 interact with?
- Same concept as HA-Swi4 but now ie. HA-HCM1
HA-HCM1 DNA
Generate Graph
of Data
Cy5= Labeled DNA interacted
with HA-tagged proteins
Cy3= Labeled DNA did not
interact with HA-tagged proteins
A-Untagged vs untagged control
B- HCM1 vs untagged
C- TOS4 vs untagged
D- Yhp1 vs untagged
Functional Categories of the 9 Transcription Factors
SBF ⇒ 9 transcription factors ⇒ target genes → categorized
Subcategories
White category: Chromosome
structure/segregation
Red category: DNA
synthesis/repair
Enrichment/association of target
genes with each subcategory
Major Processes Controlled by the Target Genes
Polarized growth/budding
DNA synthesis and repair
Mitotic functions
Energy generation
Proteolysis
Cell cycle regulation
http://www.biology-pages.info/Y/Yeast.html
Major Transcription Factors Involved in Budding
MIPS categories
● Polarized Growth (Blue)
● Cell Wall (Green)
Budding
●
●
Polarized Growth
○ Cytoskeleton polarization
Cell Wall
○ Cell wall & plasma membrane
components
○ Synthesis (carbohydrate metabolism
and transport)
http://www.genetics.org/content/191/2/347
Transcription Factors Involved in DNA Synthesis
Significance: ensure the
fidelity of DNA replication
Category: DNA
synthesis/replication (red)
Synthesis, repair,
chromosome structure
Transcription Factors Involved in Mitosis
Mitosis
Spindle pole body duplication, assembly, and positioning
DNA replication
Chromosome
structure
organization
Cytoskeleton
organization and
transport
Nuclear division
and migration
Chromosome
segregation
Budding
http://www.shmoop.com/cell-cycle/mitosis.html
cytokinesis
Transcription Factors Involved in Energy
Generation
Amino acid metabolism
Mitochondrial functions
Transcription Factors Involved in Cell Cycle
The appropriate cyclins
Regulation
must be present to
activate a certain
phase of the cell
cycle
Regulation of cyclin
concentration is
called cell cycle
regulation (dark blue
category)
http://mol-biol4masters.masters.grkraj.org/html/Cell_Cycle_And_Its_Regulation.htm
+ proteolysis
All 9 transcription factors in this study,
except Tye7, regulates cell cycle
All involved in proteolysis
Transcriptional Network
Huge network, significant number of genes, diverse and distinct functions
Hierarchical network, one transcription factor regulating the others
Important role of SBF and MBF: activate the transcriptional cascade
Significance
G1/S transition and the transcriptional
network regulating the start of cell
cycle
Interactions
Upstream, downstream
Feedbacks
Checkpoints - cancer
Homologous genes in human
3 of Hcm1 targets
https://www.studyblue.com/notes/note/n/11-cell-cycle/deck/12636531
http://www.indicasat.org.pa/publicaciones/
Improvements / Limitations
Improvement/Further Analysis:
Knockout experiment against SBF
pMBP1 in the PCR Assay
Negative control but showed up in
Swi4-HA IP DNA and did not show
up/faint in WCE DNA
Possible explanations: purification
problem (lane 1), non-specific probe
→ non-specific staining (lane 2), PCR
amplification (lane 3)
Suggested Readings
Kisseleva-Romanova, E., Lopreiato, R., Baudin-Baillieu, A., Rousselle, J.C., Ilan,
L., Hofmann, K., Namane, A., Mann, C., Libri, D. 2006. Yeast homolog of a
cancer-testis antigen defines a new transcription complex. EMBO J. 25, 3576-85.
Newly isolated yeast gene, cell cycle and polarized cell growth,
homologous to a human gene related to ovarian and testis cancer.
Sources
1. http://blog.addgene.org/plasmids-101-mammalian-vectors
2. https://en.wikipedia.org/wiki/ChIP-on-chip#/media/File:ChIP-on-chip_wet-lab.png
3. http://learn.genetics.utah.edu/content/labs/microarray/
4. http://biology-pictures.blogspot.com/2011/10/budding-yeast-picture.html
5. http://www2.le.ac.uk/departments/genetics/vgec/schoolscolleges/topics/cellcycle-mitosismeiosis
6. http://www.shmoop.com/cell-cycle/mitosis.html
7. http://www.biology-pages.info/Y/Yeast.html
8. http://mol-biol4masters.masters.grkraj.org/html/Cell_Cycle_And_Its_Regulation.htm
9. https://www.studyblue.com/notes/note/n/11-cell-cycle/deck/12636531
10.http://www.indicasat.org.pa/publicaciones/
The end
Thank you for listening!