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A cellular memory of development history generates phenotypic diversity in c.

elegans Notes

Results and Discussion

Global Differences in Gene Expression


 Assessed difference in global gene expression between wildtype (never enter dauer)
and postdauer (one day)
 Found that expression of 1181 and 946 genes were significantly upregulated and
downregulated, respectively in postdauer
 Expression ranged from 8-fold decrease to 5-fold increase
 Techniques – protocol for precise regulation of dauer entry and exit, microarray
hybridization experiments, qRT-PCR
 Affect genes include those predicted to encode proteins involved in cell cycle regulation,
transcription, phosphorylation and dephosphorylation, metabolism, reproduction, and
G protein-coupled receptor signaling
 Large percentage of genes found were associated with reproduction
o 23% of genes identified as sperm-enriched was significantly downregulated in
postdauer
o Expression of 65 of 88 predicted major sperm cytoskeleton protein (msp) genes
was downregulated in postdauer
o Passage through dauer shown to increase male survival and outcrossing rates
 Changes in gene expression may contribute to altered life history traits such as
reproduction in postdauer adults
 Examined 2127 genes characterized in dauer in postdauer animals, based on expression
in control, dauer, an postdauer and categorized into four groups
o Group 1 - similar expression levels in dauer and postdauer; indicates that
expression levels were altered during dauer and stably maintained after
o Group 2 – similar in dauer and control but were altered (either up or down) in
postdauer animals; indicates that altered expression of gene set in postdauer
adults was likely to occur following exit from dauer stage
o Group 3 – genes where expression levels either up or down regulated in both
dauer and postdauer but further up or down regulated in postdauer
o Group 4 – expression levels were distinct and uncorrelated in control, dauer, or
postdauer, indicating that regulatory mechanisms are present in dauer and in
postdauer

Chromatin Modifications in Dauer and Postdauer Animals Compared to Control Animals


 Expression of chromatin-associated genes such as histones are altered in dauer stage
 Found genes associated with nucleosome assembly and chromatin remodeling in gene
set where expression is altered in postdauer animals
 Techniques – ChIP-Seq, qPCR
 ChIP with antibodies against H3 to assess nucleosome density and against histone
modifications associated with euchromatin and heterochromatin
o Euchromatin – H3K4me3 and H4ac
o Heterochromatin – H3K9me3 and H3K27me3
 Correlated histone modifications in gene regulatory and coding sequences with overall
expression levels in control and postdauer animals
o Due to trans-splicing, took average of number of ChIP-Seq reads for each histone
modification 2kb up and downstream of translation initiation sites; then
compared histone modification with gene expression levels
 Nucleosome occupancy and overall histone H3 levels were similar in control and
postdauer regardless of gene expression
o Suggests that histone modifications rather than nucleosome content may be
correlated with gene activity
 Found H3K4me3 and H4ac modifications decreased genome-wide in postdauer animals,
especially in highly expressed genes, despite similar gene expression
 H3K27me3 and H3K9me3 levels were similar in control and postdauer
 Further examined observed changes in chromatin state in postdauer by selecting genes
with highest expressed category and quantified levels of H3K4me3 and H4ac
modifications in coding and regulatory sequences
o Average levels of both modifications were decreased in up/downstream
sequences of examined genes
o Verified chromatin modification level changes using ChIP
o Quantified histone modification levels in regulatory sequences using qPCR
 Fund decrease in H3K4me3 and H4ac levels in sequences examined
 Results imply that genome-wide chromatin modification in postdauer is marked distinct
from control animals
 Next, examined the 2127 genes that had altered expression in postdauer and compared
the chromatin states associated with these genes with control
o Found similar trends to genome-wide experiments; both H3K4me3 and H4ac
modifications were decreased in highest expressed gene category in postdauer
o Mainly observed in upregulated genes
 Levels of both modifications had positive correlation with gene activity in
up/downregulated genes subsets
 No correlation between fold change in expression and chromatin modification between
postdauer and control
 Results indicate that gene subsets whose expression is altered in postdauer is subject to
overall developmental history-dependent chromatin modification changes similar to
genome-wide modification changes
 Changes in expression of individual genes could be due to additional genetic or
epigenetic mechanisms at the local level
 Then investigate whether mutations in chromatin remodeling genes affect postdauer
expression changes
o Look at highly downregulated msp-64 and upregulated choline/carnitine o-
acyltransferase gene W03F9.4 in mutants with defective chromatin remodeling
pathways
 Found that although expression changes in both genes in subset of mutant postdauer
and WT postdauer, mutations in additional chromatin remodeling genes either
decreased, abolished, or reversed expected levels of up/downregulation in postdauer
o Each mutation had different effects on msp-64 and W03F9.4
 Implies that remodeling of chromatin architecture plays role in establishment or
maintenance of postdauer expression changes and that different mechanisms may
affect expression of different gene sets
 Determined if altered gene expression and chromatin modification profiles were
associated with altered phenotypes in postdauer, by quantifying adult life span and
brood sizes of postdauer and control
 Mean adult life span of postdauer was significantly extended compared to control
 Postdauer had more progeny
 Mutations in the subset of chromatin remodeling genes abolished brood size differences
between control and postdauer
 Embryonic lethality and adult survival rates were similar between control and postdauer
in all genetic backgrounds
 Indicate that spending one day in dauer is sufficient to after physiology and life history
traits in postdauer; further supports that changes in chromatin state may be causal to
phenotypic differences
 To determine if increased spermatogenesis accounted for increased brood size in
postdauer, quantified time course of progeny production by control and postdauer
o Brood sizes in self-fertilizing hermaphrodite are limited by number of sperm
produced
 Found that postdauer produced more progeny on later days suggesting that total sperm
number may be increased in postdauer animals
 Results indicate that the downregulated sperm-enriched genes in postdauer may be
compensated by other mechanisms, resulting in larger brood size