The Aurora A-HP1? pathway regulates gene expression and mitosis in cells from the sperm lineage

Function of HP1? phosphorylation during mitotic cell division in male germ cells

Unexpectedly, phenotypic examinations in mice carrying a gene trap that disrupts the
HP1? gene, initially generated for experiments that did not seek to necessarily shed
light into reproductive biology, demonstrated that alterations in this chromatin protein
result in azoospermia 8]. However, there is a paucity of data on how this protein is regulated to support
the development and maturation of the sperm lineage. Recent studies in our laboratory
have demonstrated in somatic cells that HP1? phosphorylation at Ser⁸³ is catalyzed by Aurora A 10], a kinase that plays a significant role in supporting proliferation throughout development
17]. Thus, to begin filling this important knowledge gap, we initially studied the localization
specifically of the Ser⁸³-phosphorylated form of HP1? in mouse testis sections using immunohistochemistry.
Notably, we found that P-Ser⁸³-HP1? localizes most strongly within cells along the basement membrane of seminiferous
tubules, where mitotic spermatogonia and pre-leptotene spermatocytes reside (Fig. 1a). In order to determine whether P-Ser⁸³-HP1? could be detected in later stages of spermatogenesis, we performed higher resolution
imaging on mature sperm by electron microscopy, which revealed that this phosphorylated
subpopulation of HP1? is stored primarily in the centriole and mid-piece region at
the base of the sperm nucleus (Fig. 1b and c). This localization was further confirmed by immunofluorescence, which clearly
showed that the signal for phosphorylated HP1? was not localized within the sperm
nuclei, as shown by DAPI counterstain, but rather coincided with the mid-piece (Fig. 1d). In addition, P-Ser⁸³-HP1? colocalized with Centrin-2, a structural component of the centrosome, which
is located within the sperm mid-piece (Fig. 1e). This localization of P-Ser⁸³-HP1? is congruent with the fact that the kinase responsible for this modification,
namely Aurora A, has also been shown to be localized to centrioles 18].

Fig. 1. P-S83-HP1? is present in testicular tissue and spermatozoa. a. P-S83-HP1? in normal mouse testicular tissue. Immunohistochemistry of P-S83-HP1?
(left panel) reveals that the cell population near the basal lamina, typically undergoing
high levels of proliferation, is the most highly P-S83-HP1? positive. Right panel
demonstrates negative control of immunohistochemistry staining with secondary antibody
only. Scale bar represents 50 ?M. b and c. P-S83-HP1? localization in spermatozoa by electron microscopy. Immunogold electron
microscopy depicting mature human spermatozoa. P-S83-HP1? was concentrated in the
centriole (b) and mid-piece (c, arrow) of the spermatozoa. d and e. P-S83-HP1? localization spermatozoa by immunofluorescence. Representative image
for protein localization of human spermatozoa with immunofluorescence. Localization
of P-S83-HP1? is observed adjacent to the sperm nuclei (counterstained with DAPI in
d), corresponding to the mid-piece of mature human sperm. This signal also co-localizes
with centrin-2 (red in e), structural component of the centrosome located in the mid-piece

We subsequently asked whether the role of P-Ser⁸³-HP1? is an exclusive feature of male cell division or it is also found in oocytes
and, after fertilization, during mitosis in early embryos. Interestingly, we found
that M2 oocytes did not demonstrate localization of P-Ser⁸³-HP1? at spindle poles (Fig. 2a). Similarly, newly fertilized embryos, which do not yet exhibit pronuclear syngamy
(Day 0), did not display P-Ser⁸³-HP1? localization to euchromatin as previously described in somatic cells 9]. However, embryos undergoing the first mitotic cell division transitioning from pronuclear
syngamy (Day 0.5) to the 2-cell stage (Day 1) demonstrated localization of P-Ser⁸³-HP1? at the spindle poles, colocalizing with ?-tubulin. This event coincides with
mouse early embryonic genome activation 19]. This localization was maintained in mitotic cells from the first division through
the late blastocyst stage. Day 3 early blastocysts (approximately 32 cells) and day
5 late blastocysts (100 cells) also demonstrated localization of P-Ser⁸³-HP1? to its euchromatic location (Fig. 2a). These experiments were complemented with quantitative PCR, which demonstrated
that HP1? transcript was 18.1-fold higher (±6.3-fold) in spermatozoa and 3.6-fold higher (±0.03-fold)
in M2 oocytes than in the newly fertilized, pronuclear embryo (day 0) when normalized
to GAPDH levels (Fig. 2b). There was a 11.8-fold increase (±0.01-fold) in HP1? expression at the day 0.5 pronuclear syngamy stage compared with the day 0 pronuclear
embryo before syngamy and a 168.3-fold increase (±1.4-fold) at the 2-cell stage (day
1). However, HP1? expression levels decreased after this point through the early blastocyst (day 3,
125.1?±?0.75-fold compared to day 0) and late blastocyst (day 5, 14.7?±?0.07-fold
compared to day 0) stages (Fig. 2b). Thus, combined, these results suggest that phosphorylation of HP1? at Ser⁸³ plays a key role in mitotic cell division in the male germ line and early embryonic
genome activation, which is likely contributed by the sperm.

Fig. 2. Spatiotemporal phosphorylation and expression of HP1? increases during early embryonic
development. a. Immunofluorescence of P-S83-HP1? in mouse oocytes and embryos. Immunofluorescence
depicting P-S83-HP1? (green), ? Tubulin (red) and DAPI DNA staining (blue). P-S83-HP1?
colocalizes to centrosomes at the time of mouse early embryonic genome activation
(Day 0.5; arrow) and maintains this location through the remainder of mitotic divisions.
After syngamy this protein then localizes to its euchromatic location during interphase
in cells in the preimplantation embryo.b. HP1? mRNA expression during early embryonic development. mRNA from mouse oocytes
and embryos were analyzed by Q-PCR. Relative expression was normalized using ??Ct
for HP1? levels to GAPDH. Lowest value in each analysis (Day 0) was normalized as 1 with fold changes depicted
in logarithmic units on the Y-axis. Error bars represent S.E.M. Time course of embryos
post HCG injection was Day 0 (18 hrs), Day 0.5 (29.5 hrs), Day 1 (42 hrs), Day 3 (90 hrs),
Day 5 (138 hrs)

Genetic inactivation of HP1? in cultured male germ cell lines leads to mitotic aberrations

Using immortalized mouse male germ cell lines (GC1 and GC2), well-suited models for
functional studies on the sperm lineage corresponding to the pre-meiotic spermatogenic
cell population 11], 13], for which we observed the highest P-Ser⁸³-HP1? levels, we first measured the protein levels of both HP1? and its phosphorylated
Ser⁸³ form by western blot. Fig. 3a demonstrates that indeed these proteins can be readily detected, leading us to proceed
with experiments based on the genetic inactivation of HP1?. For this purpose, we proceeded
to knockdown HP1? using stable lentiviral shRNA, which achieved an approximate 90 %
reduction in protein levels as demonstrated by western blot analyses (Fig. 3b). In addition, examination of knockdown cells by immunofluorescence further confirmed
decrease of HP1? and P-Ser⁸³-HP1? staining in cells transfected with HP1?-specific shRNA. More importantly, phenotypic
examination of these cells determined that the genetic inactivation of HP1? results
in mitotic defects, which include centrosome abnormalities, multipolar spindles, and
unorganized chromosomes (Fig. 3c) compared with control. Quantitative analyses revealed that these abnormalities
were significantly induced by HP1? knockdown (26.5 %) when compared to scrambled control
shRNA (shCTRL) cells (Fig. 3d; 1.5 %; n?=?200 in each group). Concordantly, the shHP1? cell population also demonstrated
decreased cell division compared to shCTRL cells, as measured by mitotic index assay
(Fig. 3e; 78.1 %?±?1.3 % of shCTRL). Thus, these results demonstrate that normal levels of
HP1? are necessary to maintain normal mitotic cell division in pre-meiotic cells from
the sperm lineage, a finding that is congruent with our immunohistochemical observations.

Fig. 3. Knockdown of HP1? in germ cells results in mitotic abnormalities. a. Levels of P-S83-HP1? in GC1 and GC2 cell lines. High levels of HP1? and its phosphorylated
Ser83 form are found in the cell lines, GC1 and GC2, by Western Blot. b. Knockdown of HP1? in GC1 cells. shRNA-mediated knockdown of HP1? results in reduction of HP1? protein levels as shown by western blot. ?-tubulin
is used as loading control. c. Immunofluorescence of HP1? knockdown in GC1 cells. Immunofluorescence of control cells (shCTRL) shows colocalization
of P-S83-HP1? staining in green and ?-tubulin in red, creating a yellow signal in
the overlay. DNA is counterstained with DAPI (blue). ShRNA knockdown of HP1? abolishes P-S83-HP1? staining (loss of green signal) and results in centrosomal
abnormalities compared with control cells. A representative shHP1? cell is shown where
centrosomes are labeled by ?-tubulin staining (red) to demonstrate aberrant spindle
pole number and localization during mitosis. d. Quantification of mitotic abnormalities. Quantification reveals a significantly
high rate of centrosomal abnormalities in the shHP1? cells vs shCTRL, 26.5 % and 1.5 %
respectively. e. HP1? knockdown in GC1 cells results in decreased cell division. Mitotic index assay confirms
that shHP1? cells have decreased cell division compared to shCTRL cells (78.1 %?±?1.3 %;
normalized to shCTRL), likely as a result of these mitotic abnormalities

HP1? regulates gene expression networks that are key for supporting normal spermatogenesis

Since the major biochemical function of HP1? is to regulate gene expression, we next
examined the effects of this protein on genome-wide expression profiling that may
influence spermatogenesis. For this purpose, we utilized our GC1 cell line stably
expressing an HP1? shRNA knockdown construct. When compared to control shRNA cells
(Fig. 4a), the genetic inactivation of HP1? resulted in 273 genes being significantly upregulated
or downregulated. Further processing of this data using a Gene Ontology (GO) ANOVA
analysis demonstrated that HP1? knockdown significantly impacted biological processes
involved in sperm development (p??0.05) (Fig. 4b). This relationship was apparent by the differential regulation of gene targets
involved in both mitosis and meiosis-related processes such as those ontologically-related
to the regulation of the cell cycle and mitosis (Additional file 2: Table S1, Fig. 4b). To validate these results, we used real time quantitative PCR to measure the expression
of a subset of spermatogenesis targets identified as significant by the Affymetrix
analysis (Fig. 4c). These experiments sought to validate changes in the expression of genes with the
following associated processes: meiosis (Stag3), spermatogenesis (Brd2), cell motility (Il16), response to stress (Carhsp1, Sod2, Ahr, Hmox1), among others (Srpk1). Complementary, Ingenuity-based analysis showed that the top-scoring gene networks
differentially modified by HP1? knockdown were related to cellular development, gene
expression, and cell cycle. For instance, a representative example of this type of
gene networks, shown in Fig. 4d, pertains to regulation of the Wnt signaling pathway, which has widely been implicated
in the promotion of proliferation and unipotent properties of spermatogonial stem
cells 20]. Therefore, we conclude that our genome-wide data obtained though the knockdown of
HP1? in male germ cell lines is congruent with a role for this protein in male germ
cell division, as supported by both our immunochemical analyses and mechanistic cell
biological experiments.

Fig. 4. Knockdown of HP1? in male germ cells impacts processes related to mitosis and meiosis. a. Affymetrix whole genome gene expression analysis was performed on GC1 HP1? knockdown cells compared to scrambled shRNA control cells. 273 genes targets are
significantly (fold change ±1.25, p??0.005) activated or repressed in the absence
of HP1?. b. Gene Ontology (GO) ANOVA analysis of the 273 targets was performed and revealed
significant (p??0.05) enrichment of mitosis and meiosis associated processes, as
well as processes involved in differentiation. c. qPCR validation of a subset of identified knockdown targets with known function
in male fertility is shown. Fold change of shHP1? compared to shCTRL expression is
represented on a scale of ±2 and shown next to the corresponding Affymetrix data.
d. The top-scoring Ingenuity-based network analysis network is significantly (p??0.05)
associated with cellular development, gene expression, and cell cycle

Phosphorylation at Ser⁸³ plays a role in HP1?-mediated regulation of spermatogenesis-associated gene expression
networks

To characterize the relationship of HP1? phosphorylation on the regulation of genes
identified by HP1? knockdown, we performed a rescue experiment by expressing wild
type HP1? or phosphorylation mutants. Toward this end, we utilized our GC1-HP1? knockdown
cells and transduced them with adenoviral vectors expressing empty vector (EV), wild
type HP1? (WT-HP1?), the non-phosphorylatable mutant (HP1?-S83A), or a phosphomimetic
form of Ser⁸³-HP1? (HP1?-S83D) for Affymetrix whole genome gene expression analysis (Additional
file 3: Table S6). Genes that were not significantly regulated by WT-HP1?, HP1?-S83A, or
HP1?-S83D (p??0.95, fold change ±2, adjusted to EV expression) were compared to genes
significantly altered in the presence of HP1? knockdown (p??0.05, fold change ±1.25). Rescue was a priori defined as a significant reversal in expression of the gene loci identified by HP1?
knockdown in the presence of either wild type or phospho-mutant HP1? (S83A or S83D).
Of the 273 genes affected by HP1? knockdown identified in the previous experiment
(Fig. 4a), 79 genes were not rescued by WT-HP1? or either mutant (Additional file 4: Table S2), which suggests that their expression is not directly modulated by HP1?
or is an artifact of the gene knockdown. Expression of the phosphomimetic (S83D) and
the non-phosphorylatable (S83A) forms rescued 77 genes (39.69 %; Additional file 4: Table S2), indicating that a significant portion of HP1? function in these cells
is dependent on phosphorylation. Notably, both mutants altered expression of a large
subset of genes not identified in the knockdown rescue unique from wild type HP1?
overexpression, suggesting that mutation of the serine 83 site and altered phosphorylation
status may possess profound pathway disruption effects. Additionally, 117 genes were
rescued by WT-HP1? (43 %; Additional file 4: Table S2). As the serine 83 site on the wild type HP1? molecule is intact, the dependency
of phosphorylation on the rescue of these genes is possible but indeterminate. From
these data, we conclude that the expression of a subset of spermatogenesis-associated
genes identified by HP1? knockdown requires not only the expression but also the phosphorylation
of this protein for their transcriptional control.

To gain better insight into how HP1? phosphorylation status affects spermatogenesis-associated
gene networks, we performed gene enrichment ontological analysis of gene targets rescued
by WT and the phosphorylation mutants (Fig. 5a-c). Accordingly, we found that WT-HP1? rescued genes involved in various aspects
of mitosis, including spindle checkpoint, protein localization to the centrosome,
centriole replication, and centrosome duplication (Fig. 5a). Various processes related to morphogenesis were significantly enriched, such as
meiosis, apoptosis, and cellular differentiation. Processes rescued by the S83A mutant,
but not the S83D mutant, included G1/S regulation, as well as processes involved in
delays or arrest of mitosis, indicating a requirement for HP1? dephosphorylation during
these events (Fig. 5b). Targets rescued by the S83D mutant, which were surrogates for genes which their
expression requires HP1? phosphorylation, participate in mitotic G1/S checkpoint as
well as cellular differentiation (Fig. 5c). A number of signaling cascades displayed enrichment with both mutants (Additional
file 5: Table S3, Additional file 6: Table S4, Additional file 7: Table S5), including Wnt, RAS, ERK, MAPK, and TNF, signifying a requirement for
HP1? phosphorylation in the regulation of gene networks that support differentiation,
growth, and survival processes during spermatogenesis 20]–24]. Taken together, these results support a role for HP1? in cell cycle processes intrinsic
to the expansion and differentiation of germ progenitor cells in a manner that is
highly dependent on the Ser⁸³ phosphorylation status of this protein.

Fig. 5. Rescue of mitosis and meiosis processes mediated by HP1? is dependent on its phosphorylation
status. a-c. Gene Ontology (GO) ANOVA analysis of rescued targets was performed and revealed
significant (p??0.05) enrichment of mitosis and meiosis associated processes, as
well as processes involved in cellular expansion and differentiation for (a) HP1?, (b) HP1? and HP1?-S83A but not HP1?-S83D or (c) HP1? and HP1?-S83D but not HP1?-S83A. Targets, rescued by the wild type protein but
not one of the mutants, indicate targets explicitly dependent on HP1? phosphorylation
or dephosphorylation