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Session 4: The Epigenome of Fertile Human Sperm.

Most of the genome of a fertile spermatozoan is packaged in protamine.

However, a small subset of genes remain bound to nucleosomes., and many regulate
development and/or transcription.

Nuclear Compaction During Spermiogenesis in the Guinea Pig-

During This Process Histones are Replaced by Protamines
But in sperm of fertile men, 0.3 to 10% of histones remain. These histones, their methylation
and the genes with which they are associate are important to embryo development.

David Phillips

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Sperm are
transcriptionally
silent. But the
distribution
and modification of sperm
histones affects the
expression of a gene’s
paternal allele in the
embryo. DNA that is associated
with retained histones
can be digested with
Micrococcal nuclease.

DNA that is associated

with protamine is not
digested with
Micrococcal nuclease.
After digestion of
Chromatin with nuclease
DNA in histone-rich areas of (cleaving at sequence
the sperm genome is packaged associated with histones)
in nucleosomes. Protamine-coated
DNA can be recovered by
Centrifugation.

Chromatin remodeling and epigenetic modifications in human sperm. DNA methylation is the first line of epigenetic modification of chromatin
through methylation of position of cytosines found in CpG dinucleotides. An intermediate step in demethylation is the formation of 5-
hydroxymethylcytosine residues, which are also observed in mature sperm. DNA is bound to histone octamers with unique modifications that
present a second level of regulation of gene transcription. Most histones are removed from the elongating spermatid and replaced with
protamines that result in a higher order of DNA packaging and silence gene expression. Retained histones are interspersed between protamine
toroids and may be bound to matrix attachment regions, which facilitates replication of loop domains in the embryo.
Douglas T. Carrell Epigenetics of the male gamete Fertility and Sterility, Volume 97, Issue 2, 2012, 267–274

Saher Sue Hammoud, David A. Nix, Haiying Zhang, Jahnvi Purwar, Douglas T. Carrell &
Bradley R. Cairns (2009) Distinctive chromatin in human sperm packages genes for
embryo development. Nature 460: 473-489.

Hypothesis: In fertile human sperm, histones and unmethylated CpG islands are
associated with specific classes of genes.

Specific Experimental questions

1. In sperm are specific genes bound by nucleosomes or are nucleosomes randomly

distributed?

2. What genes and what regions of these genes are bound by transcriptionally
permissive methylated histone 3 (H3K4me3, H3K4me2) or by transcriptionally repressive
methylated histone 3 (H3K27me3)? Are any genes associated with both H3K4me3 and
H3K27me3 and thus, potentially poised for transcription in the embryo?

3. Which gene promoters are bound by histones and not by protamines? Which
promoters are associated with H3K4me3, H3K4me2 (transcriptionally permissive), with
H3K27me3 (transcriptionally repressive) or with protamine?

4. Which gene promoters contain methylated CpG islands? Which don’t?

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get the sperm; isolate the DNA

Experimental Approach:
1. Digest sperm chromatin with micronuclease. Pellet protamine-bound DNA by centrifugation.
The micronuclease could cle
ave the DNA around the nucl
What genes are associate w 2. Use chromatin immunoprecipitation to isolate nucleosomes with the following histone 3 eosomes but it could not cl
ith the nucleasomes? modifications: eave the protamine-bound DNA.
Uae the antibody that can Dimethylated lysine 4 or trimethylated lysine 4 (transcriptionally permissive)
directly interact with the m Trimethylated lysine 27 (transcriptionally repressive)
odified nucleosomes. OR
Through isolate the nuc Separate DNA enriched in 5’methyl-CpG islands from DNA that is not methylated.
leosomes-antibody co
mplex we can see the 3. Identify genes or gene promoters enriched in isolated nucleosomes and in immunoprecipitated
modification of the nucl
eosomes and which kin chromatin. you have to have a computer program that can organize the data to make sense to the aggregate
d of gene associate wit
h them. 4. Bioinformatic analysis of data to determine if genes in specific pathways or with specific functions
are associated with nucleosomes and specific histone modifications.

think of the biochemical pathways; up-regulate/down-regulate

you want ot organize the genes and know the bichemical pathways/reactions relate to them.

Two different methods were used to identify genes or gene

promoters enriched in nucleosomes in immunoprecipitated chromatin or
in other DNA fractions:

1. Next generation DNA sequencing: Counts the relative number of

times a specific DNA sequence is present in a sample.

2. Oligonucleotide array analysis.

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gene chip: Oligonucleotide Array Analysis

oligonucleotides: complementary
to the specific genes and sequenc
es

the test sample and the control sampl

e are labled by the two kindso f fluros
cences, it the fluroscemce in the hole
s are more red, the more genes(high
concentration) in the test samples ot
herwise, the more gene in the control
samples.

the machine can scan the chips and ex

am the flurosences to tell you the rele
cative gene contention in the samples.

Sources: Affymetrix, Bioinformatics-SA,

https://www.ebi.ac.uk/training

Gene Ontology (GO) Analysis

What are the functions of genes associated with sperm nucleosomes and with
specific histone modifications?
In what pathways, processes or cellular components do these genes act?

• Captures and organizes information about each of the

genes encoded by the DNA in a sample1.
• Assigns genes to specific molecular pathways, biological
processes or to specific cellular components (e.g.
mitochondria).

• Determines if a sample is significantly enriched in genes

in specific pathways, processes or that encode specific
cellular components. This analysis allows one to make
functional inferences.
1 Go analysis is also used to evaluate transcriptomes, proteomes and metabolomes.

8 those genes that are bound to the nuc

losomes highly enriched relative to t
he whole genome. GO analysis: The gen
e you caputures in the nucleosomes in
clude in the biological in pathways.
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Identifying Genes Enriched In an Experimental Sample

Experimental Sample Reference Sample

the experimen
tal samples ar What are the types
e the genes th Each colored circle = of GO terms?
at bind ot the Experimental
specific gene. GO contains three k
nucleosome sample contains ey categories: cellul
only a fraction of Size of circle = no. of
the total genome. copies of that gene
ar component (CC;
In sample. where gene product
s are active), molec
Genes with same color ular function (MF; th
are described by the
same GO term.
e biological function
of gene or gene pro
duct) and biological
Experimental Sample= Gene copies in reference sample= process (BP; pathw
(A) DNA isolated from nucleosomes (A) Total sperm DNA ays or larger proces
(B) DNA isolated from nucleosomes with specific (B) DNA in nucleosomes ses that multiple gen
histone modifications.
(C) DNA enriched in 5’-methyl guanine.
e products involved i
(C) Total genomic DNA.
n).
Use GO analysis to identify genes with a specific GO term that are overrepresented in the
Experimental Sample.
9 the total DNA containing in two samples are the same but the subtypes are different as
Figure shows.

Making Functional Inferences By Use of Gene Ontology Analysis

Sort genes enriched in experimental sample by GO term.

Determine if GO terms are significantly overrepresented in the list of enriched genes:

Experimental Sample (No. of enriched genes with Specific GO term /

Total Number of Genes Detected)

VS.

Reference Sample (Total No. Genes with Specific

GO term / Total Number of Genes Detected.)

Make functional inferences based on overrepresentation of GO terms.

In this study: What early embryonic processes are affected by the paternal genome?

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doi: 10.1038/nature08162 SUPPLEMENTARY INFORMATION

Supplemental Figure 2

Experimental Designs 1 2 3
Nature 460: 473, 2009 Histone Analysis
Histone Analysis
Using Microarray
DNA Methylation Analysis

Using Illumina GAII Donor 2

And
Experimental
. Questions: Sperm from Donor 4
Donors Pooled sperm Donor 1
1 donor
1,2,3,4
In sperm, what genes From 4 donors
1 are enriched in the Extract Sperm
nucleosome fraction? Extract Sperm
DNA
Sequential MNase
What are the histone 3 Sequential MNase
DNA
Digestion
Digestion
modifications of these
nucleosomes? Cy 3 Gel Purify Shear,
Isolate
Supplemental Figure Legends:
Mononucleosome Mononucleosomal Mononuc. Protamine-
Denature
In sperm, which Pool DNA Pool Bound
DNA, and

2 gene promoters are Supplemental Fig. 1: Composition of

DNA (By
Incubate with
anti- 5meC
Cy 5human sperm chromatin. a, Quantifying histone
centrifugation)
enriched in nucleosomes?
content
Purify
of primary fibroblast or human sperm cells by immunoblot analysis with the H3C
What are the histone 3 DNA ChIPs Cy 5
terminus antibody. ChIPsb, Sequential digestion of sperm chromatin with increasing
TH2B
modifications of these H3K4me3 H3K4me3 Elute Antibody-
MeDIP
concentrations
Illumina GAII of micrococal nuclease (MNase) releases
H3K4me2 H3K4me2 mononucleosomes (lanes
Bound DNA 1 and
Eluate
Input
nucleosomes? Sequencing H3K27me3 H3K27me3
2), whereas protamine-packaged chromatin
Histone-
Enriched
resists MNase (lane 6). c, Characterizing the
Purify
mononucleosome fraction DNA releasedPromoters
into the Cy MNase
3 supernatant pool from panelCyb.
Protamine 5
d, Gel-Cy 3
In sperm, which gene
3 purified
Histone- mononucleosomal DNA usedH2Bfor array hybridization or sequencing. e,
Cy 5 Localization
Illumina GAII
promoters do not contain Quantification
Enriched
of the amount of histone
Sequencing
H2A
H3 H4 released by MNase treatment. Supernatants were
Loci
methylated CpG islands? pooled. Here, cell equivalents were loaded in each lane; 4% of the total supernatant or
protamine Loci pellet. The Histones
with Modified gel was subjected to immunoblotting and quantified on a Typhoon Me

(Amersham). f, Western analysis, involving titrations

Promoters for bulk levels of H3K4me3,GC AT CG GC GC
with Modified
Histone Or TH2B
H3K4me2, H3K27me3 in primary fibroblast cells and mature sperm cells. QuanitationMe
by Typhoon (Amersham) reveals that sperm bear ~4% of the histone H3 present inMethylated a
Promoters
primary
Analysisfibroblast.
of Whole Genome Analysis of Gene Promoters
Supplemental Fig. 2: Schematic representation of experimental procedures. Two fertile
The more times a DNA donors were used for methylation studies, one donor (D1) was used for all histone
sequence is detected in these modifications studied on the arrays. A pool of fertile donors were utilized for
experiments, the more mononucleosome localization and characterization and to extend the analysis genome-
enriched the sequence is in wide using Illumina GAII.
the sample
Supplemental Fig. 3: Chromatin attributes of the HOXA, HOXB, and HOXC loci.
Histone enrichment (red bars), or histone modifications (H3K4me3 array results (ruby),
11 H3K4me3 sequencing normalized difference scores (grey), H3K27me3 sequencing
normalized difference scores (teal blue) or H3K4me2 (violet)). The y-axis is the signal
intensity (log2 for array data, or normalized difference score for Illumina GAII
sequencing) and the x-axis is the annotated physical map (HG18). a, The HOXA locus.
b, The HOXC locus c, The HOXB locus.

Supplemental Fig. 4: Certain self-renewal genes as well as genes required for embryonic
development generally lack DNA methylation and are bivalent. a, SOX2 and FOXD3 are
member of the pluripotency netwok. SOX2 is demethylated and characterized by the
1 In fertile human sperm
presence what are
of H3K4me3 andthe functions
H3K27me3, ofFOXD3
whereas genesisthat are enriched
hypermethylated in
near their
nucleosomes, transcription
and in start sites. OCT4 and NANOG are also hypermethylated (Supplementary
histones with a transcriptionally permissive
Fig. 10c). b, Genes involved in embryonic development are typically DNA modification
(H3K4me3) and/or a transcriptionally
hypomethylated, repressive
and have high levels modification
of H3K4me2/3 and H3K27me3 (H3K27me3)
around their start
sites The red asterisks indicate the region amplified for bisulfite sequencing in
(N=1arrays,
Supplementary Fig. 10. The y-axis is the signal intensity (log2 for ChIP-chip pool) or
normalized difference for Illumina GAII sequencing score) and the x-axis is the
annotated physical map (HG18).
www.nature.com/nature H3K4me3: 2
Nucleosome: Go Terms Permits
Supplemental Fig 5: Developmental and signaling factors are deficient in DNAtranscription
146 bp DNA,H2A, Genesmethylation, 30 signaling
enriched inalthough Notch pathway members are hypermethylated. a, Notch
H2B, H3 and H4
sperm nucleosomes are DKK1 (hypomethylated) and NOTCH1 (hypermethylated).
pathway members, 210 b, FGF
described
364
Enrichment of genes

by GO terms:
the nucleosome
with GO terms:

177
552 1207
Number of genes/ 60
GO term
5 Go Terms
38 14
59
18
H3K27me3:
54 Represses
transcription
139

309

Note preponderance of GO terms are for

CHIP = chromatin immunoprecipitation
transcription and development

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enriched in nucleosomes
2
(1 donor analyzed)

Go Terms H3K4me2
Promoters of genes
associated with sperm permits
nucleosomes are transcription
described by GO terms:

Chip
H3K4me2
H3K4me3 Go terms

H3K4me3
Cy 5- and Cy 3-labeld DNA are permits
added to the same array chip. Ratio transcription.
(Cy 5 to Cy 3; mononuclear DNA or
Immunoprecipitated DNA to total
nucleosomal DNA) defines the
enrichment of a DNA sequence.
As with the whole genome analysis (1) most GO terms are
for transcription and development

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In human sperm what are the functions of genes in promoters with hypomethylated DNA?
3

22
DNA is immunoprecipitated
with an antibody to 102
Number of genes/
5-methy cytosine. Test for GO term
83
enrichment of sequences
with unmethylated 95
CpG dinucleotides. 75

Preponderance of GO terms for genes with

hypomethylated DNA are for
transcription and development

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Summary of Ontology Analysis of the Genes

in Human Sperm That Are
Enriched in Nucleosomes.

The human genome contains approximately 21,000 protein-coding genes.

In fertile sperm, 4,556 genes were associated with histones.

1,683 of these genes are grouped into Gene Ontology Categories associated
with development and/or transcriptional regulation.

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both methylation; block binding Genes Poised for Transcription Are Associated with Histones with Both
of RNA polymerase; there is no Transcriptionally Permissive and Repressive Methylation
t methylation of the CpG island

Blocks
they 27 demethylation, the gene can transcript.

Demethylation of H3K27
permits gene expression

Blocks

Modified from: Development 141: 3619, 2014

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The epigenome of some genes in a normal sperm genome

identify them as poised for rapid initiation of transcription.

Chromatin modifications of poised genes are characterized by:

1. CpG islands in the genes’ promoters are not methylated.

2. Some of the histones bound to these genes have transcriptionally
both permissive (e.g.H3K4me2) and
transcriptionally repressive modifications (e.g. H3K27me2).
4. In poised genes, demethylation of lysine 27 of histone 3 generates
chromatin that is transcriptionally permissive.
5. Many poised genes play important roles in development.

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In Human Sperm Some Genes That Are Essential for Early Embryonic Development
Have Chromatin Modifications Characteristic of Genes Poised for Transcription.
DNA in promoters is hypomethylated
Score: Statistical representation of enrichment of sequence

Sox 2: Required for self-renewing FOXD3: Required for

replication of the inner cell mass trophoblast development

Note hypomethylation
of sperm DNA.

H3K27 H3K27
me3 me3
H3K4 H3K4
me3 me3
Promoter/Enhancer Promoter/
Enhancer

Score = enrichment of
3’ end of gene’s coding sequence 5’ end of gene’s coding sequence sequence in immuno-
precipitate of chromatin.

H3K27Me3: transcriptionally repressive H3K4 me2: transcriptionally permissive

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Both Sox 2 and FOXD3 are

SOX 2 expression is essential for
essential for very early
formation of the inner cell mass
embryonic development.

FOXD3 is essential for

for development of
trophoblasts

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SUMMARY of Hammoud et al.

Nucleosomes in human sperm are associated with genes associated with development
and/or transcription.

Many of those genes are bound by histones with transcriptionally permissive

modifications.

Others (e.g. SOX2 and FOXD3) are bound by histones with both transcriptionally
permissive and transcriptionally repressive modifications. The promoters of those
genes do not contain 5-methy cytosine residues.
Thus, they are poised for transcription in the embryo.

Conclusion: The sperm epigenome potentially affects embryonic development.

Studies of the effects on embryonic development of manipulating the

epigenomes of mouse sperm support this conclusion.

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M. Oikawa et. Al. Epigenetic homogeneity in histone methylation underlies

sperm programming for embryonic transcription. Nature Communications
(2020)11: 3491.

How similar are the epigenomes of each spermatozoan

in an ejaculate of a fertile man?

Hypothesis: Sperm in fertile human ejaculate have highly similar

epigenomes. The regulatory regions of a few genes are almost uniformly
bound by Histone 3 lysine 4 trimethyl or by histone 3 lysine 27 trimethyl.
Most of these genes are involved in development of the early embryo.

The experiments in this publication quantify the percentage of sperm that

carry an epigenetic mark at a specific region of the sperm genome.

The experimental protocols used by Hammoud et. al., did generate such
quantitative data.

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0.2 of the genome that responsible for the high histone density of the genome.

Quantitative Analysis of the Distribution of Histone-Bound DNA in Human Sperm

Which DNA Sequences in the Human Sperm Genome are Bound by H3K4me3 or H3K27me3?
What are the Densities of H3K4me3 and H3K27me3 at those sequences
What fraction of sperm have those modified histones at that site?

HMD = Histone methylation density

0.2% of the Genome of Each Human Sperm is Associated with High Histone Density

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Are Transcriptional Regulatory Regions of Some Genes Bound by Histones?

Are histones bound to these regions in all sperm?

Structure of a Protein Coding Gene

Transcription Start Site = TSS

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H3K4me3 in genomic areas that overlap the transcription start sites of 1570 genes.

Each row = 1 gene.

Data are clustered
for similar histone
methylation
densities.

Near homogeneous H3K4Me3

across a broad region that
overlaps the TSS of 1570 genes

TSS= transcription start site

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Quantitative Analysis of the Distribution of Histone-Bound DNA in Human Sperm

Which Gene TSSs Are Associated with Peaks of H3K4me3 or H3K27me3?
What are the Densities of H3K4me3 and H3K27me3 at Those TSSs?
What fraction of sperm have those modified histones at That TSS?

H3K27me3 peak
H3K4me3

Transcription Start Site

H3K27me3
130 gene TSSs are associated with a peak of H3K4me3

320 genes TSSs are associated with a peak of H3K27me3

DNA of those 320 genes is not methylated. These
genes are poised for transcription.
Many regulate development.

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An important conclusion of M. Oikawa et. al.

In almost every fertile human sperm, regulatory regions of few genes are bound by
histone 3 lysine4 trimethyl and/or histone 3 lysine 27 trimethyl. A significant percentage
of these genes encode intrinsic regulators of development.

The epigenome determines which of a sperm’s genes are the first to be expressed in
an embryo. The proper, early expression of these genes may be essential for normal
embryonic development.

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