Download Breanna Perreault D145 Presentation 2/23/17 Background

Breanna Perreault
D145 Presentation
2/23/17
Background: Epigenetics
Why are we studying?
- Traditional Genetics Insufficient Explanation
What is Epigenetics?
- A change in phenotype without a change in genotype;
alteration of gene expression without alteration of endogenous DNA code
Epigenetics Vocabulary
If you have a smartphone, I recommend taking a picture of this slide now :)
Histone Methylation: Methylation on Histones
DNA Methylation: Direct Methylation on DNA, especially on CpGs
CpGs: Consecutive C and G nucleotides, sequence that can be directly methylated
CpG Islands: Areas with very high CG content, can help regulate transcription
Intergenerational: All “existent” at time of subject’s examination
Transgenerational: All following generations yet to be created
Knowns Before Entering The Experiment
Father’s experiences seems to have an effect on progeny
-Past Studies
Methylated CpGs not responsible for implied heritability in some studies
- CpG methylation heritable
Overview:
Variant Histone (not DNA) Methylation and Sperm RNA linked to altered
transgenerational gene expression
H3K4me2 (Dimethylated Histones on transcription factor binding regions)on
developmental genes reduced by expression of KDM1A histone lysine 4 demethylase
Paternal Epigenetic Inheritance Effects in development and fitness lasted two
generations, including those without mutant genotype (heritable)
Intergenerational / Transgenerational
Background: Paternal Inheritance
This study focused on Spermatogenesis from Primordial Germ Cells (or PGCs)
- Rapid cell division
- Highly compacted nucleus- protamine
- Low histone levels
Promoter regions of housekeeping genes : histone variant H3.3, H3K4 di- and trimethylation.
Developmental regulatory genes: H3.3, H3.1 &H3.2 variants & trimethylation of H3K27
Creation of Transgenic Animals
Microinjection into Embryos
P EF1a - Translation elongation factor EF1A
(gonad specific, germ not somatic)
IRES - internal ribosome entry site
EGFP - Green Flourescent Protein
Confirming Transgene
Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end
labeling (TUNEL). TdT enzyme, adds dUTPs that are secondarily labeled with a marker.
B RT PCR
C In situ hybridization; mRNA (blue) in spermatogonia (SG), spermatocytes (SC), and
round spermatids (RS)
Experimental Breeding Crosses
Phenotypic Results
Phenotypic Results
Claims Normalization TG4 (subjective)
Mortality
Results
Proposed Mechanism of Variable Deformities
Mortalities and abnormalities non- mendelian
-KDM1A is active in spermatogenesis; diploid cells.
-Cellular bridges from incomplete mitotic and meiotic divisions;
transgene in 50% of spermatids but all haploid spermatids affected
Histone Localization Analysis
Which genes are affected?
1. DTT treatment - remove protamine
2. Micrococcal Nuclease digestion
3. Chromatin immunoprecipitation (ChIP) with H3K4me2specific antibodies
4. ChIP-seq ( DNA to protein, next gen, massively parallel)
ChIP-seq
Location of Demethylation
(Chip Seq Results)
KDM1A to transcription start sites with H3.3 and H3K4me2
Therefore with high CpG content
-Gene ontology
H3K4me2 involved in morphogenesis, patterning, vasculature development, cartilage
development, and catabolic and metabolic processes
KDM1A preferentially alters offspring development.
Gene Ontology
Pdpk1 = embryogenesis, growth, and nervous system development
E2F6 = skeletal defects
Gsc = craniofacial abnormalities
F3 Progeny has reduced H3K4me2
Proposed Mechanism:
TSS (transcription start site )regions with reduced H3K4me2 levels are strongly
enriched for H3.3
High levels of H3.3 = high nucleosome turnover at CGIs in spermatids
KDM1A cannot maintain H3K4me2 state during nucleosome turnover in spermatids of
TG3 males.
F3 Progeny has reduced H3K4me2
Similar demethylation methylation to start, Mature TG changes
MATURE Spermatozoa
PRE-Spermatazoa
Any other way Non-TG offspring got
phenotypic aberrations?
Considering methylation mechanism of
phenotypic aberrations in Non-TG offspring
- Target selection: CpGs differentially methylated in TG3 sperm & C57BL/6 controls
- Quantitative MassARRAY (with bisulfite)
- RBBS (Reduced representation bisulfite sequencing)
Result: Not methylation
MassARRAY
iPLEX assay
Extension primer
RBBS
(Reduced representation bisulfite sequencing)
RNA products of Overexpression of KDM1A
Late
Affymetrix GeneChip ST2.0 Array for RNA content
Affymetrix gene chip
Early
Overlapping Mutant Genotypes
Findings
Critique
Purpose of study is often explained as environmental, but methods not environmental.
Distinction between RNA or altered Histones as reason for epigenetic inheritance
Use of human gene to create the experimental variable in the mouse
Phenotypic analysis of the transgenic pups not quantitative
Borrowed Data
Bibliography & Further Reading
This article:
Keith Siklenka1,*, Serap Erkek2,3,*,†, Maren Godmann4,‡, Romain Lambrot4, Serge McGraw5, Christine Lafleur4, Tamara Cohen4,
Jianguo Xia4,9, Matthew Suderman7, Michael Hallett8, Jacquetta Trasler5,6, Antoine H. F. M. Peters2,3,*,§, Sarah Kimmins1,4,*,§1Dep, Keith
Siklenka, Serap Erkek, Maren Godmann, Romain Lambrot, Serge McGraw, Christine Lafleur, Tamara Cohen, Jianguo Xia, Matthew Suderman,
Michael Hallett, Jacquetta Trasler, Antoine H. F. M. Peters, Sarah Kimmins, and Published Online08 Oct 2015. "Disruption of Histone Methylation in
Developing Sperm Impairs Offspring Health Transgenerationally." Disruption of Histone Methylation in Developing Sperm Impairs Offspring Health
Transgenerationally | Science. N.p., n.d. Web. 23 Feb. 2017.
http://science.sciencemag.org/content/350/6261/aab2006/tab-pdf
Article on the histone analysis method:
Caglayan, A. O., Comu, S., Baranoski, J. F., Parman, Y., Kaymakçalan, H., Akgumus, G. T., et al. (2015). NGLY1 mutation causes
neuromotor impairment, intellectual disability, and neuropathy. Eur J Med Genet, 58(1), 39-43.
http://www.nature.com/nprot/journal/v8/n12/full/nprot.2013.145.html
Article on an interesting paternal epigentic phenomenon
B. G. Dias, K. J. Ressler, Parental olfactory experience influences behavior and neural structure in subsequent generations. Nat. Neurosci.
17, 89–96 (2014)
http://www.nature.com/neuro/journal/v17/n1/full/nn.3594.html
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