MCB Accepts, published online ahead of print on 13 October 2014 Mol. Cell. Biol. doi:10.1128/MCB.00794-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Mice Without MacroH2A Histone Variants
2 3 John R. Pehrson1,2, Lakshmi N. Changolkar1,3, Carl Costanzi1,4 and N. Adrian Leu1,5
4 5 6
1
7
Philadelphia, PA 19104, USA
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania,
8 9
3
Current address: Department of Pathology and Lab Medicine, University of Pennsylvania,
10
Philadelphia PA 19104, USA
11
4
Current address: Western Maine Health, Norway ME 04268, USA
12
5
Current address: Department of Human Genetics, The University of Utah, Salt Lake City UT
13
84112, USA
14 15
2
Corresponding author: [email protected]
16 17
Running title: Mice Without MacroH2A Histone Variants
18 19
Key words: chromatin; histone variants; gene regulation; metabolism
20 21
Introduction, Results and Discussion word count (characters excluding spaces): 29,919
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Material and Methods word count (characters excluding spaces): 6592
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1
24
Abstract
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MacroH2A core histone variants have a unique structure that includes a C-terminal nonhistone
26
domain. They are highly conserved in vertebrates, and are thought to regulate gene expression.
27
However the nature of genes regulated by macroH2As and their biological significance remain
28
unclear. Here we examine macroH2A function in vivo by knocking out both macroH2A1 and
29
macroH2A2 in the mouse. While macroH2As are not required for early development, the absence
30
of macroH2As impaired prenatal and postnatal growth, and can significantly reduce reproductive
31
efficiency. The distribution of macroH2A.1- and macroH2A.2-containing nucleosomes shows
32
substantial overlap, as do their effects on gene expression. Our studies in fetal and adult liver
33
indicate that macroH2As can exert large positive or negative effects on gene expression, with
34
macroH2A.1 and macroH2A.2 acting synergistically on the expression of some genes and
35
apparently having opposing effects on others. These effects are very specific, and in the adult liver
36
preferentially involve genes related to lipid metabolism including the leptin receptor. MacroH2A-
37
dependent gene regulation changes substantially in postnatal development and can be strongly
38
affected by fasting. We propose that macroH2As produce adaptive changes to gene expression,
39
which in the liver focus on metabolism.
40
2
41
Introduction
42
MacroH2A core histone variants have a unique structure in which an N-terminal H2A domain is
43
connected to a C-terminal nonhistone domain [1]. The H2A domain can substitute for conventional
44
H2A in the nucleosome, and we estimated that ~ 1 in 30 nucleosomes contains a macroH2A in
45
adult rat liver [1, 2], an organ with relatively high macroH2A content. Most of the nonhistone
46
region consists of an evolutionarily conserved domain [3] called a macrodomain. Macrodomains
47
are found in a variety of proteins and as stand-alone proteins in many bacteria [3, 4]. Some, but not
48
all macrodomains bind ADP-ribose with high affinity [5, 6].
49
MacroH2As show a complex distribution in metazoan organisms [7]. The basal metazoan
50
species Trichoplax adhaerens and the sponge Amphimedon queenslandica have a macroH2A gene,
51
as does Hydra magnipapillata, clearly indicating its presence in early metazoan evolution. While
52
some other invertebrate genomes contain a macroH2A gene, many complete or nearly complete
53
invertebrate genomes lack macroH2A; e.g., macroH2A is present in a sea urchin
54
(Strongylocentrotus purpuratus), a tick (Ixodes scapularis) and an annelid (Capitella teleta), but is
55
absent in sequenced insects, a nematode (Caenorhabditis elegans) and a tunicate (Ciona
56
intestinalis). Two highly conserved macroH2A genes, macroH2A1 and macroH2A2, are found in
57
mammalian genomes, and clearly homologous genes can be found in birds, reptiles and fish. To
58
our knowledge, macroH2A genes have not been detected in fungal, protozoan, or plant genomes.
59
These findings indicate that macroH2As first appeared in early metazoan evolution, were lost in
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some invertebrate lineages, but were strongly retained in higher vertebrate species.
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In mice, humans and many other vertebrates, macroH2A1 transcripts are alternately spliced
62
to produce two macroH2A.1 variants, macroH2A.1.1 and macroH2A.1.2 that differ in an ~30
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amino acid region of the macrodomain [1, 8]. MacroH2A2 appears to encode a single protein,
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macroH2A.2, that has the same size and general structure as the macroH2A.1 variants, but is only
3
65
about 70% identical in primary structure [9, 10]. The macrodomain of macroH2A.1.1 binds ADP-
66
ribose [6], although the functional significance of this interaction for macroH2A1.1 nucleosomes is
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unclear. There is no identified small molecule that binds the macrodomains of macroH2A.1.2 or
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macroH2A.2. MacroH2As are present in all organs we have examined including liver, brain, testis,
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thymus, kidney and adrenal gland. However, the macroH2A content and composition of different
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tissues can be significantly different [9, 11]. For example, macroH2A.1.1 appears to be present
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mainly in non-proliferating cells of differentiated tissues [11, 12].
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Although it is generally believed that regulation of gene expression is an important role for
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macroH2As, there is no consensus on the nature of the genes that are targeted or the biological
74
significance of this regulation. A study of gene expression in MACROH2A1 knockdown MCF-7
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cells indicated that macroH2A.1 impacts the expression of many genes, with 19% of 97 genes
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examined showing significantly altered expression [14]. A role in gene silencing is suggested by
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the preferential localization of macroH2As to domains of transcriptionally silent heterochromatin
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including the inactive X chromosome [9, 10, 15], pericentromeric heterochromatin in some cells
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[13, 16], the XY-body of spermatocytes [17] and transcriptionally silent senescence-associated
80
heterochromatic foci [18]. Cell-type specific macroH2A-dependent silencing of IL-8 was observed
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in studies of cultured cell lines [19], suggesting that macroH2As could be important for
82
establishing or maintaining cell-type specific patterns of gene expression. Other studies indicate
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that macroH2As have a critical role in early development and differentiation. A knockdown of
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macroH2A2 in developing zebrafish embryos led to severe developmental defects [20], and some
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studies of embryonic stem (ES) cells found that knockdown of macroH2A1 and/or macroH2A2
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impaired the expression of differentiation genes and the inactivation of pluripotency genes during
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in vitro differentiation [21, 22]. In contrast, our studies of macroH2A1 knockout (KO) mice
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showed no obvious defects in development or X-inactivation [23]. Changes in liver gene
4
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expression were seen for a few genes that functionally cluster in the area of metabolism. Most of
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these genes showed macroH2A.1 enrichment [24], suggesting that they are direct targets of
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macroH2A.1 nucleosomes. Our results seemed to be inconsistent with the studies that showed
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dramatic effects on early development and stem cell differentiation. One potential contributor to
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this apparent inconsistency is macroH2A.2, which could have overlapping functions with
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macroH2A.1 variants and mask significant aspects of macroH2A function in macroH2A1 KO
95
mice. To address this possibility and expand our understanding of macroH2A function in vivo, we
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knocked out macroH2A2 and generated mice that lack both macroH2A.1 and macroH2A.2 histone
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variants.
98 99
Materials and Methods
100
Mice
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The University of Pennsylvania Institutional Animal Care and Use Committee approved all animal
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protocols. Mice were maintained on a 12 hr day/night cycle with the lights coming on at 8 AM. Mice
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used for organ harvest were euthanized with CO2 between 9 and 11 AM. For DEXA body composition
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analyses the mice were anesthetized (100mg/Kg Ketamine + 10mg/kg Xylazine) and scanned (PIXImus,
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GE Lunar).
106 107
MacroH2A KO mice
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MacroH2A1 KO mice inbred in either the C57Bl/6 or 129/S6 (Taconic) background for at least 10
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generations were produced as previously described [23]. Genotyping was done with a small piece of the
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tail that was digested with proteinase K. Primers: F1 - TCTTTGCAAGGGTCAGACG; R1 –
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ACTGGAGACAGCGCATTACC; R2 – CAACATAACCACCATGATCTCG. Reactions used Taq
5
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polymerase (New England Biolabs), annealing temperature 55o C (normal allele 148 bp, KO allele 238
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bp).
114
The macroH2A2 (H2afy2) KO targeting vector was based on a ~6.8 kb BglII-KpnI fragment of
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mouse genomic DNA cloned into the pPNT vector [25] (Fig. 1S). The Neo cassette was flanked by
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LoxP recombination sites and inserted into an EaeI site located less than 300 bp upstream of exon 2. A
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third LoxP site was inserted into an EaeI site located less than 150 bp downstream of exon 2. The
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targeting vector was electroporated into V6.5 ES cells [26]. ES cells positive for homologous
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recombination by southern blotting (Fig. 1S) were injected into blastocysts and the chimeric males were
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mated to obtain germ line transmission. Recombinant mice were bred with an EIIa Cre mouse (Jackson
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Laboratories) and offspring were screened by PCR to identify mice carrying KO alleles. The
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macroH2A2 KO allele was backcrossed into the C57Bl/6 and 129/S6 backgrounds for 10 or more
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generations. Genotyping primers: AF1 - CCCAGCCAATCCCAAGTATGAGG; CR1 -
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CCCCAACTCACTCCATATGCAGC. Tail DNA PCR reactions used AccuPrime Taq polymerase
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(Invitrogen), annealing temperature 55o C (normal allele 895 bp, KO allele 320 bp), (Fig. 1S).
126 127
Microarrays
128
Wild-type and macroH2A KO females were super-ovulated and mated with wild-type or macroH2A KO
129
males respectively. Wild-type and KO embryos were collected, mixed together and transferred to wild-
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type pseudo-pregnant females. Livers were collected from 18.5 d.p.c. fetuses. In studies involving adult
131
mice, wild-type and KO mice were raised together following weaning, and were euthanized at 2 months
132
of age at ~ 10 AM. The liver was immediately dissected and frozen in liquid nitrogen. Total RNA was
133
isolated using Trizol (Invitrogen).
134
Labeled cDNA was prepared from 250 ng of total RNA using the Ambion WT Expression Kit
135
(Life Technologies). Labeled cDNA (5.5 g) was hybridized to Mouse Gene 2.0 ST GeneChips (adult
6
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livers) or Mouse Gene 1.0 ST GeneChips (fetal livers) (Affymetrix Inc., Santa Clara CA). The
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microarrays were stained with streptavidin-phycoerythrin. A GeneChip 3000 7G scanner was used to
138
collect the fluorescence signal.
139
Affymetrix Command Console and Expression Console were used to quantitate expression levels
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for targeted genes; default values provided by Affymetrix were applied. Border pixels were removed,
141
and the average intensity of pixels within the 75th percentile was computed for each probe. The average
142
of the lowest 2% of probe intensities occurring in each microarray sector was subtracted as background
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from all features in that sector. Affymetrix cel files (containing probe intensities) were imported into
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Partek Genomics Suite (v6.6, Partek, Inc., St. Louis, MO) where RMA normalization was applied
145
yielding log2-transformed intensities for all Transcript IDs. Normalized intensities were exported from
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Partek into R, where Significance Analysis of Microarrays (SAM, using the samr v2.0 package, [27])
147
was applied, yielding a fold change and q-value (False Discovery Rate, FDR) for each Transcript ID.
148
The normalized data and cel files were deposited in the NCBI GEO database, accession number
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GSE57089.
150 151
Real-time PCR
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Total RNA was prepared from mouse liver using Trizol (Invitrogen). We synthesized cDNA using
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random hexamer primers and M-MuLV Reverse Transcriptase (RNase H–) (New England Biolabs)
154
under standard conditions. Real-time PCR was performed and quantitated using the LightCycler System
155
(Roche Applied Science) as previously described [28]. Primer sequences and annealing temperature are
156
listed in Table 5S.
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Purification of macroH2A nucleosomes
159
MacroH2A-containing mono- and oligonucleosomes were purified from bulk chromatin by thio-affinity
160
chromatography as described [28]. Briefly, fifty mixed gender 18.5-d.p.c. fetal mouse livers were very
161
gently homogenized using a Potter Elvehjem tissue homogenizer, and nuclei were purified by
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ultracentrifugation through a sucrose cushion; fetal liver nuclei must be handled with care to avoid
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aggregation. An H1-stripped chromatin fraction was prepared and digested to mono- and
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oligonucleosomes. H1 removal was done at 60 mM NaCl instead of the 50 mM previously used, which
165
improved removal. Nuclease digestions used recombinant micrococcal nuclease (New England Biolabs),
166
and the protease inhibitor cocktail cOmplete (Roche) was used throughout the procedure. The
167
solubilized nucleosomes were first passed through a column of Activated Thiol Sepharose (GE
168
Healthcare Life Sciences), which was connected in tandem to a column of Thiopropyl Sepharose (GE
169
Healthcare Life Sciences). The columns were disconnected, and washed and eluted as previously
170
described [28], except we first washed the Activated Thiol Sepharose column with 0.5 M NaCl, 1 mM
171
EDTA, 10 mM Tris pH 7.5 prior to elution with a 100 mM -mercaptoethanol, 100 mM NaCl, 1 mM
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EDTA, 10 mM Tris pH 7.5. This elution procedure was also used for the Thiopropyl Sepharose column;
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the 0.5 M NaCl wash removes nucleosomes bound to the column through cysteine-containing
174
nonhistone proteins. The same thio-affinity procedure was used for a control experiment done with ~50
175
double KO 18.5-d.p.c. livers.
176 177
High-throughput sequencing and mapping of sequences
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Mononucleosomal DNA was purified from an agarose gel, and the DNA ends repaired using standard
179
procedures. An A was added to the 3’ ends using Klenow (exo-), and Illumina multiplex adaptors were
180
ligated to the fragments following standard procedures. Ligated fragments were gel purified and
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amplified using Illumina multiplex primers. Libraries were sequenced to 50 bp using an Illumina HiSeq
8
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2000 sequencer. Reads were aligned to the mouse genome (release mm9) using Bowtie, allowing up to 3
183
mismatches using the flags '-v 3 -k 1 -m 1 --best –strata’. The sequences were deposited in the NCBI
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GEO database, accession number GSE57089. For the comparison of the macroH2A.1 track (Thiopropyl-
185
eluted) to the macroH2A.2 track (Activated Thiol-eluted) (Fig. 4C), the macroH2A over input (starting
186
material) ratios were computed by tabulating the expected center of fragments corresponding to each
187
read, then assigning them to sliding-window bins. The log2 ratio was computed with a pseudocount of 1
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added to both macroH2A and input read totals. Bins that had 0 macroH2A and input reads were
189
discarded. The log2 ratios were normalized by subtracting the genome-wide median log2 ratio.
190 191
Western blots
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Proteins were separated by SDS gel electrophoresis, transferred to polyvinylidene difluoride membranes
193
[11], and probed with rabbit polyclonal antibodies raised against the nonhistone regions of rat
194
macroH2A1.1, 1.2 and human macroH2A.2 [15]. The signal was detected by chemiluminescence. The
195
blot in Fig. 1 used a low bisacrylamide SDS polyacrylamide gel, which allows the resolution of all three
196
macroH2A variants [9]. The blots in Figs. 3, 4 and 2S used a gel with a standard
197
acrylamide:bisacrylamide ratio of 37.5:1, which does not resolve macroH2A.1.1 from macroH2A.1.2.
198 199
Results
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Knockout of macroH2A2
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We knocked out macroH2A2 by removing the second exon (see Methods and Fig. 1S). This exon
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contains the initiator methionine and encodes the first 57 amino acids of the histone region of
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macroH2A.2. As in the case of the knockout of macroH2A1, the macroH2A2 KO did not produce
204
any obvious developmental defects or reproductive problems. We kept a small group of
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macroH2A2 KO and wild-type mice in a mixed C57Bl/6 x 129/Sv background for more than 1.5
206
years and did not observe any obvious pathology in the KOs in comparison to controls.
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The macroH2A2 KO allele was backcrossed into the C57Bl/6 and 129/S6 (Taconic)
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backgrounds for 10 or more generations, which should eliminate most background effects except
209
for regions closely linked to macroH2A2 (H2afy2). We used an F1 (C57Bl/6 x 129/Sv) ES cell
210
line, and sequence polymorphisms around macroH2A2 showed that the KO allele is from the
211
129/Sv background. Our macroH2A1 KO allele is also in the 129/Sv background. The phenotypic
212
effects of the macroH2A double KO are different in the C57Bl/6 and 129/S6 backgrounds as
213
described below.
214 215
Normal development in the absence of macroH2A
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We were able to produce macroH2A double KO males and females, and western blotting
217
confirmed the absence of macroH2A.1 and macroH2A.2 (Fig. 1). External and internal
218
examination of late stage fetuses, newborn pups and adults did not reveal any obvious structural
219
defects associated with the absence of macroH2As. We did a histological examination of major
220
organs and tissues from two young adult double KOs and an age-matched control and did not
221
observe any obvious anomalies or pathology in the double KOs. These results show that mouse
222
development can proceed in an apparently normal fashion in the absence of macroH2A variants.
223 224
Reproductive problems of C57Bl/6 macroH2A double KOs
225
We efficiently bred macroH2A double KOs with one another in the 129/S6 background, but
226
C57Bl/6 double KOs bred very poorly with one another. In some cases the female did not appear
227
to become pregnant, while in other cases many or all of the pups died or were eaten shortly after
228
birth. Young adult C57Bl/6 double KOs typically produced offspring when bred with wild type
10
229
mice or mice expressing at least one macroH2A gene, showing that these mice are not generally
230
infertile. We believe the effects of the macroH2A double KO exacerbate reproductive and
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mothering problems that occur with wild-type C57Bl/6 mice [29]. In addition, double KOs appear
232
to be more susceptible to perinatal death (see below).
233 234
Reduced size of macroH2A double KOs
235
Double KO pups in both the 129/S6 and C57Bl/6 backgrounds were smaller on average than wild-
236
type pups and this difference persisted into adulthood. To demonstrate these effects we mixed
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wild-type and double KO pre-implantation embryos and implanted them into wild-type recipient
238
females. An experiment tracking the weights of double KO and wild-type C57Bl/6 males is shown
239
in Fig. 2, and similar results were observed with females. We used dual-energy X-ray
240
absorptiometry (DEXA) to analyze the size and body composition of 129/S6 adult double KO and
241
control males (Table 1). The double KOs weighed less and were 9% shorter in length. Most of the
242
weight difference was accounted for by reduced lean body mass. The bone mineral content, bone
243
mineral density and fat content of the double KOs were not significantly different from their wild-
244
type littermates.
245
We used C57Bl/6 mice to examine whether the absence of macroH2As reduced the size of
246
late stage fetuses. We bred double KO males with females that were heterozygous for macroH2A1
247
and KO for macroH2A2. Fetuses were collected at 17.5 and 18.5 days post coitum (d.p.c.). We
248
obtained an equal number of double KO fetuses and fetuses that were heterozygous for
249
macroH2A1 and KO for macroH2A2, but the double KO fetuses were smaller, Table 2. A similar
250
result was obtained with a group of fetuses that were produced by breeding double KO males with
251
females that were KO for macroH2A1 and heterozygous for macroH2A2, Table 2.
11
252
The reduced size of late stage double KO fetuses and the slower growth of double KO pups
253
and young adults show that the absence of macroH2As impairs pre- and post-natal growth. This
254
suggests a role for macroH2As in regulating metabolic, neurologic and/or hormonal controls of
255
growth. Our previous discovery that macroH2A.1 variants regulate metabolic genes in the liver is
256
consistent with this idea, although the absence of macroH2A.1 by itself did not have any obvious
257
effect on growth.
258 259
Increased perinatal death of C57Bl/6 macroH2A double KOs
260
Our breeding experiences found a deficiency in the number of C57Bl/6 double KOs that survived
261
to the age of weaning (3 weeks). Table 3 summarizes the results we obtained when we bred mice
262
with different combinations of macroH2A KO alleles. Overall, the number of macroH2A double
263
KOs produced was ~ 29% less than expected based on the genotypes of the parents. We observed
264
significant numbers of mice dying shortly after birth, and genotype analyses of 25 dead newborns
265
found a strong bias toward double KOs (84%). Interestingly, we did not observe a significant
266
deficiency of double KOs in our studies of 71 late stage fetuses (Table 2). Taken together these
267
results indicate that the perinatal period is the important time when macroH2A double KOs were
268
lost. More double KO females survived to weaning than males, although the difference was small;
269
this indicates that issues related to X-inactivation are unlikely to be a major factor in the loss of
270
double KO females.
271 272
Additional phenotypic effects in 129/S6 macroH2A double KO mice
273
Double KO 129/S6 mice frequently showed signs of bilaterally increased palpebral fissure in
274
comparison to controls. Histological analysis of the globes did not reveal any abnormalities and
275
histological analysis of the heads found no orbital mass or cyst that could cause a protrusion of the
12
276
globes. We conclude that this is a true macroblepharon due to an abnormally increased length of
277
the eyelid margin. Some double KOs developed eyelid inflammation - typically between 6 and 12
278
months of age - possibly secondary to eye irritation caused by the wider eyelid margin.
279
Some 129/S6 macroH2A double KO mice showed increased darkening of the hair on their
280
back. This effect appeared at ~ 6 weeks of age and was typically more evident in young adults than
281
older mice.
282 283
MacroH2A-dependent gene regulation in development - fetal liver
284
Our current hypothesis is that gene regulation is an important function of macroH2A histones. Our
285
previous studies of macroH2A1 KO mice identified genes that appear to be regulated by
286
macroH2A.1 in adult liver, but not in neonatal liver [23]. To increase our understanding of the
287
developmental aspect of macroH2A function, we used microarrays to examine gene expression in
288
129/S6 double KO fetal liver (18.5 d.p.c.); six double KOs were compared to six controls, with the
289
control livers coming from wild-type fetuses that developed together with the double KOs in wild-
290
type recipient females. The macroH2A.1 content of late fetal liver is lower than adult liver and
291
consists almost entirely of macroH2A.1.2 [11, 23]. The macroH2A.2 content of late fetal liver is
292
relatively low, but is higher than in adult liver (Fig. 3).
293
Remarkably few genes showed altered expression in our microarray analysis of double KO
294
fetal liver. Twenty genes showed a 1.5-fold or more increase in expression in the double KOs, but
295
only Npy (encodes neuropeptide Y) and Serpina7 (encodes thyroxine binding globulin) showed a
296
low false discovery rate. Pip5k1b (encodes a phosphatidylinositol-4-phosphate kinase) showed a 2-
297
fold decrease in expression with a low false discovery rate, but no other genes showed more than a
298
2-fold decrease in expression. The increased expression of Npy and Serpina7, and the decreased
299
expression of Pip5k1b were confirmed by a real-time PCR analysis of a larger group of fetal livers
13
300
(Table 4). We also used real-time PCR to examine the expression of some genes that showed a
301
smaller change in expression and a false discovery rate of
1
Mice Without MacroH2A Histone Variants
2 3 John R. Pehrson1,2, Lakshmi N. Changolkar1,3, Carl Costanzi1,4 and N. Adrian Leu1,5
4 5 6
1
7
Philadelphia, PA 19104, USA
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania,
8 9
3
Current address: Department of Pathology and Lab Medicine, University of Pennsylvania,
10
Philadelphia PA 19104, USA
11
4
Current address: Western Maine Health, Norway ME 04268, USA
12
5
Current address: Department of Human Genetics, The University of Utah, Salt Lake City UT
13
84112, USA
14 15
2
Corresponding author: [email protected]
16 17
Running title: Mice Without MacroH2A Histone Variants
18 19
Key words: chromatin; histone variants; gene regulation; metabolism
20 21
Introduction, Results and Discussion word count (characters excluding spaces): 29,919
22
Material and Methods word count (characters excluding spaces): 6592
23
1
24
Abstract
25
MacroH2A core histone variants have a unique structure that includes a C-terminal nonhistone
26
domain. They are highly conserved in vertebrates, and are thought to regulate gene expression.
27
However the nature of genes regulated by macroH2As and their biological significance remain
28
unclear. Here we examine macroH2A function in vivo by knocking out both macroH2A1 and
29
macroH2A2 in the mouse. While macroH2As are not required for early development, the absence
30
of macroH2As impaired prenatal and postnatal growth, and can significantly reduce reproductive
31
efficiency. The distribution of macroH2A.1- and macroH2A.2-containing nucleosomes shows
32
substantial overlap, as do their effects on gene expression. Our studies in fetal and adult liver
33
indicate that macroH2As can exert large positive or negative effects on gene expression, with
34
macroH2A.1 and macroH2A.2 acting synergistically on the expression of some genes and
35
apparently having opposing effects on others. These effects are very specific, and in the adult liver
36
preferentially involve genes related to lipid metabolism including the leptin receptor. MacroH2A-
37
dependent gene regulation changes substantially in postnatal development and can be strongly
38
affected by fasting. We propose that macroH2As produce adaptive changes to gene expression,
39
which in the liver focus on metabolism.
40
2
41
Introduction
42
MacroH2A core histone variants have a unique structure in which an N-terminal H2A domain is
43
connected to a C-terminal nonhistone domain [1]. The H2A domain can substitute for conventional
44
H2A in the nucleosome, and we estimated that ~ 1 in 30 nucleosomes contains a macroH2A in
45
adult rat liver [1, 2], an organ with relatively high macroH2A content. Most of the nonhistone
46
region consists of an evolutionarily conserved domain [3] called a macrodomain. Macrodomains
47
are found in a variety of proteins and as stand-alone proteins in many bacteria [3, 4]. Some, but not
48
all macrodomains bind ADP-ribose with high affinity [5, 6].
49
MacroH2As show a complex distribution in metazoan organisms [7]. The basal metazoan
50
species Trichoplax adhaerens and the sponge Amphimedon queenslandica have a macroH2A gene,
51
as does Hydra magnipapillata, clearly indicating its presence in early metazoan evolution. While
52
some other invertebrate genomes contain a macroH2A gene, many complete or nearly complete
53
invertebrate genomes lack macroH2A; e.g., macroH2A is present in a sea urchin
54
(Strongylocentrotus purpuratus), a tick (Ixodes scapularis) and an annelid (Capitella teleta), but is
55
absent in sequenced insects, a nematode (Caenorhabditis elegans) and a tunicate (Ciona
56
intestinalis). Two highly conserved macroH2A genes, macroH2A1 and macroH2A2, are found in
57
mammalian genomes, and clearly homologous genes can be found in birds, reptiles and fish. To
58
our knowledge, macroH2A genes have not been detected in fungal, protozoan, or plant genomes.
59
These findings indicate that macroH2As first appeared in early metazoan evolution, were lost in
60
some invertebrate lineages, but were strongly retained in higher vertebrate species.
61
In mice, humans and many other vertebrates, macroH2A1 transcripts are alternately spliced
62
to produce two macroH2A.1 variants, macroH2A.1.1 and macroH2A.1.2 that differ in an ~30
63
amino acid region of the macrodomain [1, 8]. MacroH2A2 appears to encode a single protein,
64
macroH2A.2, that has the same size and general structure as the macroH2A.1 variants, but is only
3
65
about 70% identical in primary structure [9, 10]. The macrodomain of macroH2A.1.1 binds ADP-
66
ribose [6], although the functional significance of this interaction for macroH2A1.1 nucleosomes is
67
unclear. There is no identified small molecule that binds the macrodomains of macroH2A.1.2 or
68
macroH2A.2. MacroH2As are present in all organs we have examined including liver, brain, testis,
69
thymus, kidney and adrenal gland. However, the macroH2A content and composition of different
70
tissues can be significantly different [9, 11]. For example, macroH2A.1.1 appears to be present
71
mainly in non-proliferating cells of differentiated tissues [11, 12].
72
Although it is generally believed that regulation of gene expression is an important role for
73
macroH2As, there is no consensus on the nature of the genes that are targeted or the biological
74
significance of this regulation. A study of gene expression in MACROH2A1 knockdown MCF-7
75
cells indicated that macroH2A.1 impacts the expression of many genes, with 19% of 97 genes
76
examined showing significantly altered expression [14]. A role in gene silencing is suggested by
77
the preferential localization of macroH2As to domains of transcriptionally silent heterochromatin
78
including the inactive X chromosome [9, 10, 15], pericentromeric heterochromatin in some cells
79
[13, 16], the XY-body of spermatocytes [17] and transcriptionally silent senescence-associated
80
heterochromatic foci [18]. Cell-type specific macroH2A-dependent silencing of IL-8 was observed
81
in studies of cultured cell lines [19], suggesting that macroH2As could be important for
82
establishing or maintaining cell-type specific patterns of gene expression. Other studies indicate
83
that macroH2As have a critical role in early development and differentiation. A knockdown of
84
macroH2A2 in developing zebrafish embryos led to severe developmental defects [20], and some
85
studies of embryonic stem (ES) cells found that knockdown of macroH2A1 and/or macroH2A2
86
impaired the expression of differentiation genes and the inactivation of pluripotency genes during
87
in vitro differentiation [21, 22]. In contrast, our studies of macroH2A1 knockout (KO) mice
88
showed no obvious defects in development or X-inactivation [23]. Changes in liver gene
4
89
expression were seen for a few genes that functionally cluster in the area of metabolism. Most of
90
these genes showed macroH2A.1 enrichment [24], suggesting that they are direct targets of
91
macroH2A.1 nucleosomes. Our results seemed to be inconsistent with the studies that showed
92
dramatic effects on early development and stem cell differentiation. One potential contributor to
93
this apparent inconsistency is macroH2A.2, which could have overlapping functions with
94
macroH2A.1 variants and mask significant aspects of macroH2A function in macroH2A1 KO
95
mice. To address this possibility and expand our understanding of macroH2A function in vivo, we
96
knocked out macroH2A2 and generated mice that lack both macroH2A.1 and macroH2A.2 histone
97
variants.
98 99
Materials and Methods
100
Mice
101
The University of Pennsylvania Institutional Animal Care and Use Committee approved all animal
102
protocols. Mice were maintained on a 12 hr day/night cycle with the lights coming on at 8 AM. Mice
103
used for organ harvest were euthanized with CO2 between 9 and 11 AM. For DEXA body composition
104
analyses the mice were anesthetized (100mg/Kg Ketamine + 10mg/kg Xylazine) and scanned (PIXImus,
105
GE Lunar).
106 107
MacroH2A KO mice
108
MacroH2A1 KO mice inbred in either the C57Bl/6 or 129/S6 (Taconic) background for at least 10
109
generations were produced as previously described [23]. Genotyping was done with a small piece of the
110
tail that was digested with proteinase K. Primers: F1 - TCTTTGCAAGGGTCAGACG; R1 –
111
ACTGGAGACAGCGCATTACC; R2 – CAACATAACCACCATGATCTCG. Reactions used Taq
5
112
polymerase (New England Biolabs), annealing temperature 55o C (normal allele 148 bp, KO allele 238
113
bp).
114
The macroH2A2 (H2afy2) KO targeting vector was based on a ~6.8 kb BglII-KpnI fragment of
115
mouse genomic DNA cloned into the pPNT vector [25] (Fig. 1S). The Neo cassette was flanked by
116
LoxP recombination sites and inserted into an EaeI site located less than 300 bp upstream of exon 2. A
117
third LoxP site was inserted into an EaeI site located less than 150 bp downstream of exon 2. The
118
targeting vector was electroporated into V6.5 ES cells [26]. ES cells positive for homologous
119
recombination by southern blotting (Fig. 1S) were injected into blastocysts and the chimeric males were
120
mated to obtain germ line transmission. Recombinant mice were bred with an EIIa Cre mouse (Jackson
121
Laboratories) and offspring were screened by PCR to identify mice carrying KO alleles. The
122
macroH2A2 KO allele was backcrossed into the C57Bl/6 and 129/S6 backgrounds for 10 or more
123
generations. Genotyping primers: AF1 - CCCAGCCAATCCCAAGTATGAGG; CR1 -
124
CCCCAACTCACTCCATATGCAGC. Tail DNA PCR reactions used AccuPrime Taq polymerase
125
(Invitrogen), annealing temperature 55o C (normal allele 895 bp, KO allele 320 bp), (Fig. 1S).
126 127
Microarrays
128
Wild-type and macroH2A KO females were super-ovulated and mated with wild-type or macroH2A KO
129
males respectively. Wild-type and KO embryos were collected, mixed together and transferred to wild-
130
type pseudo-pregnant females. Livers were collected from 18.5 d.p.c. fetuses. In studies involving adult
131
mice, wild-type and KO mice were raised together following weaning, and were euthanized at 2 months
132
of age at ~ 10 AM. The liver was immediately dissected and frozen in liquid nitrogen. Total RNA was
133
isolated using Trizol (Invitrogen).
134
Labeled cDNA was prepared from 250 ng of total RNA using the Ambion WT Expression Kit
135
(Life Technologies). Labeled cDNA (5.5 g) was hybridized to Mouse Gene 2.0 ST GeneChips (adult
6
136
livers) or Mouse Gene 1.0 ST GeneChips (fetal livers) (Affymetrix Inc., Santa Clara CA). The
137
microarrays were stained with streptavidin-phycoerythrin. A GeneChip 3000 7G scanner was used to
138
collect the fluorescence signal.
139
Affymetrix Command Console and Expression Console were used to quantitate expression levels
140
for targeted genes; default values provided by Affymetrix were applied. Border pixels were removed,
141
and the average intensity of pixels within the 75th percentile was computed for each probe. The average
142
of the lowest 2% of probe intensities occurring in each microarray sector was subtracted as background
143
from all features in that sector. Affymetrix cel files (containing probe intensities) were imported into
144
Partek Genomics Suite (v6.6, Partek, Inc., St. Louis, MO) where RMA normalization was applied
145
yielding log2-transformed intensities for all Transcript IDs. Normalized intensities were exported from
146
Partek into R, where Significance Analysis of Microarrays (SAM, using the samr v2.0 package, [27])
147
was applied, yielding a fold change and q-value (False Discovery Rate, FDR) for each Transcript ID.
148
The normalized data and cel files were deposited in the NCBI GEO database, accession number
149
GSE57089.
150 151
Real-time PCR
152
Total RNA was prepared from mouse liver using Trizol (Invitrogen). We synthesized cDNA using
153
random hexamer primers and M-MuLV Reverse Transcriptase (RNase H–) (New England Biolabs)
154
under standard conditions. Real-time PCR was performed and quantitated using the LightCycler System
155
(Roche Applied Science) as previously described [28]. Primer sequences and annealing temperature are
156
listed in Table 5S.
157
7
158
Purification of macroH2A nucleosomes
159
MacroH2A-containing mono- and oligonucleosomes were purified from bulk chromatin by thio-affinity
160
chromatography as described [28]. Briefly, fifty mixed gender 18.5-d.p.c. fetal mouse livers were very
161
gently homogenized using a Potter Elvehjem tissue homogenizer, and nuclei were purified by
162
ultracentrifugation through a sucrose cushion; fetal liver nuclei must be handled with care to avoid
163
aggregation. An H1-stripped chromatin fraction was prepared and digested to mono- and
164
oligonucleosomes. H1 removal was done at 60 mM NaCl instead of the 50 mM previously used, which
165
improved removal. Nuclease digestions used recombinant micrococcal nuclease (New England Biolabs),
166
and the protease inhibitor cocktail cOmplete (Roche) was used throughout the procedure. The
167
solubilized nucleosomes were first passed through a column of Activated Thiol Sepharose (GE
168
Healthcare Life Sciences), which was connected in tandem to a column of Thiopropyl Sepharose (GE
169
Healthcare Life Sciences). The columns were disconnected, and washed and eluted as previously
170
described [28], except we first washed the Activated Thiol Sepharose column with 0.5 M NaCl, 1 mM
171
EDTA, 10 mM Tris pH 7.5 prior to elution with a 100 mM -mercaptoethanol, 100 mM NaCl, 1 mM
172
EDTA, 10 mM Tris pH 7.5. This elution procedure was also used for the Thiopropyl Sepharose column;
173
the 0.5 M NaCl wash removes nucleosomes bound to the column through cysteine-containing
174
nonhistone proteins. The same thio-affinity procedure was used for a control experiment done with ~50
175
double KO 18.5-d.p.c. livers.
176 177
High-throughput sequencing and mapping of sequences
178
Mononucleosomal DNA was purified from an agarose gel, and the DNA ends repaired using standard
179
procedures. An A was added to the 3’ ends using Klenow (exo-), and Illumina multiplex adaptors were
180
ligated to the fragments following standard procedures. Ligated fragments were gel purified and
181
amplified using Illumina multiplex primers. Libraries were sequenced to 50 bp using an Illumina HiSeq
8
182
2000 sequencer. Reads were aligned to the mouse genome (release mm9) using Bowtie, allowing up to 3
183
mismatches using the flags '-v 3 -k 1 -m 1 --best –strata’. The sequences were deposited in the NCBI
184
GEO database, accession number GSE57089. For the comparison of the macroH2A.1 track (Thiopropyl-
185
eluted) to the macroH2A.2 track (Activated Thiol-eluted) (Fig. 4C), the macroH2A over input (starting
186
material) ratios were computed by tabulating the expected center of fragments corresponding to each
187
read, then assigning them to sliding-window bins. The log2 ratio was computed with a pseudocount of 1
188
added to both macroH2A and input read totals. Bins that had 0 macroH2A and input reads were
189
discarded. The log2 ratios were normalized by subtracting the genome-wide median log2 ratio.
190 191
Western blots
192
Proteins were separated by SDS gel electrophoresis, transferred to polyvinylidene difluoride membranes
193
[11], and probed with rabbit polyclonal antibodies raised against the nonhistone regions of rat
194
macroH2A1.1, 1.2 and human macroH2A.2 [15]. The signal was detected by chemiluminescence. The
195
blot in Fig. 1 used a low bisacrylamide SDS polyacrylamide gel, which allows the resolution of all three
196
macroH2A variants [9]. The blots in Figs. 3, 4 and 2S used a gel with a standard
197
acrylamide:bisacrylamide ratio of 37.5:1, which does not resolve macroH2A.1.1 from macroH2A.1.2.
198 199
Results
200
Knockout of macroH2A2
201
We knocked out macroH2A2 by removing the second exon (see Methods and Fig. 1S). This exon
202
contains the initiator methionine and encodes the first 57 amino acids of the histone region of
203
macroH2A.2. As in the case of the knockout of macroH2A1, the macroH2A2 KO did not produce
204
any obvious developmental defects or reproductive problems. We kept a small group of
9
205
macroH2A2 KO and wild-type mice in a mixed C57Bl/6 x 129/Sv background for more than 1.5
206
years and did not observe any obvious pathology in the KOs in comparison to controls.
207
The macroH2A2 KO allele was backcrossed into the C57Bl/6 and 129/S6 (Taconic)
208
backgrounds for 10 or more generations, which should eliminate most background effects except
209
for regions closely linked to macroH2A2 (H2afy2). We used an F1 (C57Bl/6 x 129/Sv) ES cell
210
line, and sequence polymorphisms around macroH2A2 showed that the KO allele is from the
211
129/Sv background. Our macroH2A1 KO allele is also in the 129/Sv background. The phenotypic
212
effects of the macroH2A double KO are different in the C57Bl/6 and 129/S6 backgrounds as
213
described below.
214 215
Normal development in the absence of macroH2A
216
We were able to produce macroH2A double KO males and females, and western blotting
217
confirmed the absence of macroH2A.1 and macroH2A.2 (Fig. 1). External and internal
218
examination of late stage fetuses, newborn pups and adults did not reveal any obvious structural
219
defects associated with the absence of macroH2As. We did a histological examination of major
220
organs and tissues from two young adult double KOs and an age-matched control and did not
221
observe any obvious anomalies or pathology in the double KOs. These results show that mouse
222
development can proceed in an apparently normal fashion in the absence of macroH2A variants.
223 224
Reproductive problems of C57Bl/6 macroH2A double KOs
225
We efficiently bred macroH2A double KOs with one another in the 129/S6 background, but
226
C57Bl/6 double KOs bred very poorly with one another. In some cases the female did not appear
227
to become pregnant, while in other cases many or all of the pups died or were eaten shortly after
228
birth. Young adult C57Bl/6 double KOs typically produced offspring when bred with wild type
10
229
mice or mice expressing at least one macroH2A gene, showing that these mice are not generally
230
infertile. We believe the effects of the macroH2A double KO exacerbate reproductive and
231
mothering problems that occur with wild-type C57Bl/6 mice [29]. In addition, double KOs appear
232
to be more susceptible to perinatal death (see below).
233 234
Reduced size of macroH2A double KOs
235
Double KO pups in both the 129/S6 and C57Bl/6 backgrounds were smaller on average than wild-
236
type pups and this difference persisted into adulthood. To demonstrate these effects we mixed
237
wild-type and double KO pre-implantation embryos and implanted them into wild-type recipient
238
females. An experiment tracking the weights of double KO and wild-type C57Bl/6 males is shown
239
in Fig. 2, and similar results were observed with females. We used dual-energy X-ray
240
absorptiometry (DEXA) to analyze the size and body composition of 129/S6 adult double KO and
241
control males (Table 1). The double KOs weighed less and were 9% shorter in length. Most of the
242
weight difference was accounted for by reduced lean body mass. The bone mineral content, bone
243
mineral density and fat content of the double KOs were not significantly different from their wild-
244
type littermates.
245
We used C57Bl/6 mice to examine whether the absence of macroH2As reduced the size of
246
late stage fetuses. We bred double KO males with females that were heterozygous for macroH2A1
247
and KO for macroH2A2. Fetuses were collected at 17.5 and 18.5 days post coitum (d.p.c.). We
248
obtained an equal number of double KO fetuses and fetuses that were heterozygous for
249
macroH2A1 and KO for macroH2A2, but the double KO fetuses were smaller, Table 2. A similar
250
result was obtained with a group of fetuses that were produced by breeding double KO males with
251
females that were KO for macroH2A1 and heterozygous for macroH2A2, Table 2.
11
252
The reduced size of late stage double KO fetuses and the slower growth of double KO pups
253
and young adults show that the absence of macroH2As impairs pre- and post-natal growth. This
254
suggests a role for macroH2As in regulating metabolic, neurologic and/or hormonal controls of
255
growth. Our previous discovery that macroH2A.1 variants regulate metabolic genes in the liver is
256
consistent with this idea, although the absence of macroH2A.1 by itself did not have any obvious
257
effect on growth.
258 259
Increased perinatal death of C57Bl/6 macroH2A double KOs
260
Our breeding experiences found a deficiency in the number of C57Bl/6 double KOs that survived
261
to the age of weaning (3 weeks). Table 3 summarizes the results we obtained when we bred mice
262
with different combinations of macroH2A KO alleles. Overall, the number of macroH2A double
263
KOs produced was ~ 29% less than expected based on the genotypes of the parents. We observed
264
significant numbers of mice dying shortly after birth, and genotype analyses of 25 dead newborns
265
found a strong bias toward double KOs (84%). Interestingly, we did not observe a significant
266
deficiency of double KOs in our studies of 71 late stage fetuses (Table 2). Taken together these
267
results indicate that the perinatal period is the important time when macroH2A double KOs were
268
lost. More double KO females survived to weaning than males, although the difference was small;
269
this indicates that issues related to X-inactivation are unlikely to be a major factor in the loss of
270
double KO females.
271 272
Additional phenotypic effects in 129/S6 macroH2A double KO mice
273
Double KO 129/S6 mice frequently showed signs of bilaterally increased palpebral fissure in
274
comparison to controls. Histological analysis of the globes did not reveal any abnormalities and
275
histological analysis of the heads found no orbital mass or cyst that could cause a protrusion of the
12
276
globes. We conclude that this is a true macroblepharon due to an abnormally increased length of
277
the eyelid margin. Some double KOs developed eyelid inflammation - typically between 6 and 12
278
months of age - possibly secondary to eye irritation caused by the wider eyelid margin.
279
Some 129/S6 macroH2A double KO mice showed increased darkening of the hair on their
280
back. This effect appeared at ~ 6 weeks of age and was typically more evident in young adults than
281
older mice.
282 283
MacroH2A-dependent gene regulation in development - fetal liver
284
Our current hypothesis is that gene regulation is an important function of macroH2A histones. Our
285
previous studies of macroH2A1 KO mice identified genes that appear to be regulated by
286
macroH2A.1 in adult liver, but not in neonatal liver [23]. To increase our understanding of the
287
developmental aspect of macroH2A function, we used microarrays to examine gene expression in
288
129/S6 double KO fetal liver (18.5 d.p.c.); six double KOs were compared to six controls, with the
289
control livers coming from wild-type fetuses that developed together with the double KOs in wild-
290
type recipient females. The macroH2A.1 content of late fetal liver is lower than adult liver and
291
consists almost entirely of macroH2A.1.2 [11, 23]. The macroH2A.2 content of late fetal liver is
292
relatively low, but is higher than in adult liver (Fig. 3).
293
Remarkably few genes showed altered expression in our microarray analysis of double KO
294
fetal liver. Twenty genes showed a 1.5-fold or more increase in expression in the double KOs, but
295
only Npy (encodes neuropeptide Y) and Serpina7 (encodes thyroxine binding globulin) showed a
296
low false discovery rate. Pip5k1b (encodes a phosphatidylinositol-4-phosphate kinase) showed a 2-
297
fold decrease in expression with a low false discovery rate, but no other genes showed more than a
298
2-fold decrease in expression. The increased expression of Npy and Serpina7, and the decreased
299
expression of Pip5k1b were confirmed by a real-time PCR analysis of a larger group of fetal livers
13
300
(Table 4). We also used real-time PCR to examine the expression of some genes that showed a
301
smaller change in expression and a false discovery rate of
