VAIE for Nov 30 Journal Club Piroska Oct 2017
Van Andel Educational Institute
High School Journal Club
October 31 2017
About Journal Clubs
• Participation and discussion is the key to a
good journal club
• Ideally:
• Everyone has read the paper
• Everyone participates in the discussion
• People are there because they want to be
there not because they have to
Pick the paper: Main considerations
• Does it interest you?
• Will it interest others?
• Can you make it interesting for others?
• What is the impact of the paper?
Pick the paper: Other considerations
•
Is the paper in an area you are already familiar with?
•
Will the paper allow you to learn something that you
wanted to learn?
•
Do you understand the paper well?
•
Do you think the paper is well done/written?
•
Do you agree with the conclusions?
•
Is it off the press or classic?
Read the paper
• Read all parts of the paper:
methods and materials
**supplementary
• Pay attention to:
What is the main question?
How are they addressing that question?
What techniques did they use?
How have they organized their arguments?
Copy figures
• Decide which panels and which figures are
important
• Separate panels unless they NEED to be shown
together
• It is not essential to show every piece of data or
to show the data in the same order that they do
in the paper
P element inserted
white mutant
P element mobilized
The title of a slide should always
indicate the conclusion you want your
audience to reach.
What is shown on the slide is the evidence
to support it.
Flies with Variegated Eye Phenotypes Are
Recovered after P Element Mobilization
P element inserted
white mutant
variegated
P element mobilized
Flies with Variegated Eye Phenotypes Are
Recovered using P Element Mobilization
white mutant
P element
inserted
P element mobilized
variegated
Structure the presentation
There should be a logical progression from the
main question to each sub-question to the
conclusions
For each piece of data, follow QuARC:
Question
Approach
Results (show data)
Conclusion (in the title)
(+ Connection to next question)
Experimental Approach
• Overview of the approach
• Explain any key techniques that are used
throughout
• Details about model organism
• Details about special experimental
techniques
Add background and conclusions
Once you know the logical progression of the
presentation, include relevant background for
why the experimental questions are important
Background should funnel into research
question
As part of the conclusions, a model diagram is
very useful
The Presentation
Text in red would not be part of the
presentation
I’ve included pointers and information that I
would say in words but not include on the
slide itself
For the title slide, rather than just reading the
title , summarize in lay terms the main
question or finding of the paper
You can also tell people why you chose the
paper or what is particularly interesting about it
Background/Introduction
• Follow the title slide with background
• Introduce the disease/process
• What is the gap in knowledge
• Use specific background to set up the
main question
Drosphila melanogaster
https://www.youtube.com/watch?time_continue=34&v=qDbJnFLl3kU
Why is the eye white?
Because of mutation!
Question:
Why are some cells red and others whites in this eye?
Not because of mutation!
Why are some cells red and others whites?
Scientific question:
What is the mechanism of repressing genes?
Study of gene regulation: EPIGENETICS
Epigenetics
• One genome – but many cell types
• One genome – health or disease
• Study of mitotically heritable marks that
are not encoded in DNA sequence
•
•
•
•
•
•
Condensed
Gene-poor
Repeat rich
Late replicating
Resistant to nucleases
Highly regular packaging
Heterochromatin is marked by HP1 and HP2
in salivary gland polytene chromosomes
C: chromocenter
Hypothesis:
w+ is packaged differently in facets
Packaging determines if w+ is “ON” or “OFF”
RESULTS
Question
Approach
Results
Conclusion
(+ Connection to next
question)
How did they test the hypothesis?
1. See where in the genome w+ variegates
2. Test if changing the “packaging material”
changes variegation
3. Test if changing the “packagables” affects
variegation
4. Test how well packed w+ is in variegating versus
non-variegating flies
• Question 1: Does PEV depend on genomic
position?
• Approach:
• Jump the transgene into different parts of the
genome
• Find where it landed
• See at which landing site transgene is
variegating
• Question 1: Does PEV depend on genomic
position?
• Approach:
• Jump the transgene into different parts of the
genome (transposon mutagenesis)
• Find where it landed (in situ hybridization)
• See at which landing site transgene is
variegating (look at eyes of males)
• Results: next slides
Jump the transgene into different parts of
the genome (transposon mutagenesis)
Test
Marker
Reporter
Jump the transgene into different parts of
the genome (transposon mutagenesis)
Test
Marker
Reporter
Flies with Variegated Eye Phenotypes Are
Recovered after P Element Mobilization
white mutant
P element
inserted
P element mobilized
variegated
Where did the transgenes land?
Where did the PEV transgenes land?
How do they know here the transgene landed?
peroxidase
Where did the variegating transgenes land?
They landed in known heterochromatin regions
• Question 2: Does PEV depend on the amount of
heterochromatin proteins?
• Approach: Change the level of packaging by
crossing the PEV fly to other fly strains that carry
mutation for heterochromatin proteins:
Suppressor of variegation or Su(var) mutants
• Results: next slides
PEV depends on the amount of heterochromatin proteins
except at telomeres
HP1 low
H4 Acetylation high
Packaging has a Dose Effect on Variegation
Molecular Function of Suppressors or
Variegation
Ac
HDAC
• Question 3: Does PEV depend on the amount of
heterochromatin DNA?
• Approach: Change the amount of “packagables”
by crossing to mutant fly that has altered X or Y
PEV depends on the amount of heterochromatic DNA
except at T
Less DNA
Normal
More DNA
• Question 4: Does the variegating gene packaged
more tightly?
• Approach: “Let’s use scissors”
• Treat chromatin with restriction enzyme to
test for accessibility
• Treat chromatin with micrococcal nuclease
and examine nucleosome array over
transgene.
• Compare strongly variegating fly with control fly
“Scissor” No 1
restriction enzyme
Variegating transgene is less accessible to
nuclease digestion
Heterochromatin insertion
restriction enzyme
“Scissor” No 2
Micrococcal nuclease
Variegating transgene has tighter nucleosome
structure
Electrophoresis
DNA fragment length
Micrococcal nuclease
Summary
1. P element mobilization is a good approach for studying PEV
2. Gene repression is correlated with less accessible chromatin
3. Data support the hypothesis of heterochromatin spreading
4. Gene repression at centromeres and telomeres is different
What is the molecular mechanism of
variegation?
Conclusion:
Altered chromatin packaging
plays a role in PEV
Significance
Abstract thinking lead to correct hypothesis
“Packaging” of genes is a general theme and is
important in development and disease
Follow-up works discovered the molecular nature of
Su(var)s
“Packaging” problems can be targets for cancer
therapy
Guided or spontaneous discussion
Challenge question
Criticism
• All of the experiments are done D.melanogaster
models – will these results translate to humans?
• Variegating cell is not compared to expressing
cell
• Resolution is still low
• Still open question: What determines which cell
clones repress variegating transgene?
Questions for discussion
• Are you convinced of the authors conclusions?
• What else would you like to know?
• What would be the next experiment you would
want to do?
• What are the implications for human health?
• Which techniques did they use that are novel or
potentially useful for other experiments?
Question: Why are some cells red and others whites?
Answer: Not genetic but epigenetic difference!
Send home message: ask those questions!
Extra slides for answering questions
Position Effect Variegation
Chr X
High School Journal Club
October 31 2017
About Journal Clubs
• Participation and discussion is the key to a
good journal club
• Ideally:
• Everyone has read the paper
• Everyone participates in the discussion
• People are there because they want to be
there not because they have to
Pick the paper: Main considerations
• Does it interest you?
• Will it interest others?
• Can you make it interesting for others?
• What is the impact of the paper?
Pick the paper: Other considerations
•
Is the paper in an area you are already familiar with?
•
Will the paper allow you to learn something that you
wanted to learn?
•
Do you understand the paper well?
•
Do you think the paper is well done/written?
•
Do you agree with the conclusions?
•
Is it off the press or classic?
Read the paper
• Read all parts of the paper:
methods and materials
**supplementary
• Pay attention to:
What is the main question?
How are they addressing that question?
What techniques did they use?
How have they organized their arguments?
Copy figures
• Decide which panels and which figures are
important
• Separate panels unless they NEED to be shown
together
• It is not essential to show every piece of data or
to show the data in the same order that they do
in the paper
P element inserted
white mutant
P element mobilized
The title of a slide should always
indicate the conclusion you want your
audience to reach.
What is shown on the slide is the evidence
to support it.
Flies with Variegated Eye Phenotypes Are
Recovered after P Element Mobilization
P element inserted
white mutant
variegated
P element mobilized
Flies with Variegated Eye Phenotypes Are
Recovered using P Element Mobilization
white mutant
P element
inserted
P element mobilized
variegated
Structure the presentation
There should be a logical progression from the
main question to each sub-question to the
conclusions
For each piece of data, follow QuARC:
Question
Approach
Results (show data)
Conclusion (in the title)
(+ Connection to next question)
Experimental Approach
• Overview of the approach
• Explain any key techniques that are used
throughout
• Details about model organism
• Details about special experimental
techniques
Add background and conclusions
Once you know the logical progression of the
presentation, include relevant background for
why the experimental questions are important
Background should funnel into research
question
As part of the conclusions, a model diagram is
very useful
The Presentation
Text in red would not be part of the
presentation
I’ve included pointers and information that I
would say in words but not include on the
slide itself
For the title slide, rather than just reading the
title , summarize in lay terms the main
question or finding of the paper
You can also tell people why you chose the
paper or what is particularly interesting about it
Background/Introduction
• Follow the title slide with background
• Introduce the disease/process
• What is the gap in knowledge
• Use specific background to set up the
main question
Drosphila melanogaster
https://www.youtube.com/watch?time_continue=34&v=qDbJnFLl3kU
Why is the eye white?
Because of mutation!
Question:
Why are some cells red and others whites in this eye?
Not because of mutation!
Why are some cells red and others whites?
Scientific question:
What is the mechanism of repressing genes?
Study of gene regulation: EPIGENETICS
Epigenetics
• One genome – but many cell types
• One genome – health or disease
• Study of mitotically heritable marks that
are not encoded in DNA sequence
•
•
•
•
•
•
Condensed
Gene-poor
Repeat rich
Late replicating
Resistant to nucleases
Highly regular packaging
Heterochromatin is marked by HP1 and HP2
in salivary gland polytene chromosomes
C: chromocenter
Hypothesis:
w+ is packaged differently in facets
Packaging determines if w+ is “ON” or “OFF”
RESULTS
Question
Approach
Results
Conclusion
(+ Connection to next
question)
How did they test the hypothesis?
1. See where in the genome w+ variegates
2. Test if changing the “packaging material”
changes variegation
3. Test if changing the “packagables” affects
variegation
4. Test how well packed w+ is in variegating versus
non-variegating flies
• Question 1: Does PEV depend on genomic
position?
• Approach:
• Jump the transgene into different parts of the
genome
• Find where it landed
• See at which landing site transgene is
variegating
• Question 1: Does PEV depend on genomic
position?
• Approach:
• Jump the transgene into different parts of the
genome (transposon mutagenesis)
• Find where it landed (in situ hybridization)
• See at which landing site transgene is
variegating (look at eyes of males)
• Results: next slides
Jump the transgene into different parts of
the genome (transposon mutagenesis)
Test
Marker
Reporter
Jump the transgene into different parts of
the genome (transposon mutagenesis)
Test
Marker
Reporter
Flies with Variegated Eye Phenotypes Are
Recovered after P Element Mobilization
white mutant
P element
inserted
P element mobilized
variegated
Where did the transgenes land?
Where did the PEV transgenes land?
How do they know here the transgene landed?
peroxidase
Where did the variegating transgenes land?
They landed in known heterochromatin regions
• Question 2: Does PEV depend on the amount of
heterochromatin proteins?
• Approach: Change the level of packaging by
crossing the PEV fly to other fly strains that carry
mutation for heterochromatin proteins:
Suppressor of variegation or Su(var) mutants
• Results: next slides
PEV depends on the amount of heterochromatin proteins
except at telomeres
HP1 low
H4 Acetylation high
Packaging has a Dose Effect on Variegation
Molecular Function of Suppressors or
Variegation
Ac
HDAC
• Question 3: Does PEV depend on the amount of
heterochromatin DNA?
• Approach: Change the amount of “packagables”
by crossing to mutant fly that has altered X or Y
PEV depends on the amount of heterochromatic DNA
except at T
Less DNA
Normal
More DNA
• Question 4: Does the variegating gene packaged
more tightly?
• Approach: “Let’s use scissors”
• Treat chromatin with restriction enzyme to
test for accessibility
• Treat chromatin with micrococcal nuclease
and examine nucleosome array over
transgene.
• Compare strongly variegating fly with control fly
“Scissor” No 1
restriction enzyme
Variegating transgene is less accessible to
nuclease digestion
Heterochromatin insertion
restriction enzyme
“Scissor” No 2
Micrococcal nuclease
Variegating transgene has tighter nucleosome
structure
Electrophoresis
DNA fragment length
Micrococcal nuclease
Summary
1. P element mobilization is a good approach for studying PEV
2. Gene repression is correlated with less accessible chromatin
3. Data support the hypothesis of heterochromatin spreading
4. Gene repression at centromeres and telomeres is different
What is the molecular mechanism of
variegation?
Conclusion:
Altered chromatin packaging
plays a role in PEV
Significance
Abstract thinking lead to correct hypothesis
“Packaging” of genes is a general theme and is
important in development and disease
Follow-up works discovered the molecular nature of
Su(var)s
“Packaging” problems can be targets for cancer
therapy
Guided or spontaneous discussion
Challenge question
Criticism
• All of the experiments are done D.melanogaster
models – will these results translate to humans?
• Variegating cell is not compared to expressing
cell
• Resolution is still low
• Still open question: What determines which cell
clones repress variegating transgene?
Questions for discussion
• Are you convinced of the authors conclusions?
• What else would you like to know?
• What would be the next experiment you would
want to do?
• What are the implications for human health?
• Which techniques did they use that are novel or
potentially useful for other experiments?
Question: Why are some cells red and others whites?
Answer: Not genetic but epigenetic difference!
Send home message: ask those questions!
Extra slides for answering questions
Position Effect Variegation
Chr X