Abstract
Background
KDM6A, encoding a histone demethylase, is one of the top ten mutated
epigenetic cancer genes. The effect of mutations on its structure and
function are however poorly characterized.
Methods
Database search identified nonsense and missense mutations in the
N-terminal TPR motifs and the C-terminal, catalytic JmjC domain, but
also in the intrinsically disordered region connecting both these two
well-structured domains. KDM6A variants with cancer-derived mutations
were generated using site directed mutagenesis and fused to eGFP
serving as an all-in-one affinity and fluorescence tag to study
demethylase activity by an ELISA-based assay in vitro, apoptosis by
FACS, complex assembly by Co-immunoprecipitation and localization by
microscopy in urothelial cells and apoptosis by FACS.
Results
Independent of the mutation and demethylase activity, all KDM6A
variants were detectable in the nucleus. Truncated KDM6A variants
displayed changes in complex assemblies affecting (1) known
interactions with the COMPASS complex component RBBP5 and (2) KDM6A-DNA
associated assemblies with the nuclear protein Nucleophosmin. Some
KDM6A variants induced a severe cellular phenotype characterized by
multiple acute effects on nuclear integrity, namely, release of nuclear
DNA into the cytoplasm, increased level of DNA damage indicators RAD51
and p-γH2A.X, and mitosis defects. These damaging effects were
correlated with increased cell death.
Conclusion
These observations reveal novel effects of pathogenic variants pointing
at new specific functions of KDM6A variants. The underlying mechanisms
and affected pathways have to be investigated in future research to
understand how tumor cells cope with and benefit from KDM6A
truncations.
Supplementary Information
The online version contains supplementary material available at
10.1186/s12860-021-00394-2.
Keywords: KDM6A mutations, Mitosis defects, DNA damage, Nuclear
integrity
Background
In the past decade, deep sequencing approaches have revealed mutations
in many genes encoding epigenetic modifiers, prominently KDM6A [[35]1].
KDM6A is frequently mutated in several cancer types, especially in
urothelial cancer [[36]2–[37]4], and also in the hereditary Kabuki
syndrome [[38]5]. The human H3K27-demethylase KDM6A, also referred to
as Ubiquitously transcribed Tetratricopeptide repeat, X chromosome
(UTX), specifically removes the ε-di/trimethylation on lysine 27 of
histone H3, a repressive histone modification [[39]6]. KDM6A interacts
with other chromatin-modifying protein complexes. In particular,
interactions with KMT2C/D-complexes (COMPASS) with the core components
WDR5, RBBP5, ASH2L and DPY30 [[40]7] appear to be demethylase-dependent
at bivalent H3K27me3/H3K4 domains [[41]8]. KDM6A binding at promoter
sequences of actively transcribed genes [[42]9] is mainly
demethylase-independent and involves interactions with the SWI/SNF
chromatin remodeling complex [[43]10], coactivators CBP/p300 [[44]11],
but also components of the transcription elongation machinery, like the
SUPT6H-RNA Polymerase II complex [[45]12].
The KDM6A protein has three functionally and structurally relevant
regions conserved among several vertebrates. These include the eight
N-terminal tetratricopeptide repeats (TPRs, aa 92–385 in human), which
represent a common protein interaction motif [[46]13]. Interestingly,
the human KDM6A TPR3 and TPR5 conform poorly to the consensus (1–4%) as
highlighted in Figure S[47]1, but secondary structure predictions
indicate that the essential helix-turn-helix motif is still preserved.
The well-characterized C-terminal and highly conserved, catalytically
active Jumonji C domain (JmjC) is important for histone
H3K27me2/3-recognition, binding and demethylation [[48]14]. In addition
to the JmjC core (approx. aa 1095–1260), flanking regions termed pre-
and post-JmjC are essential for its catalytic activity (approx. aa
880–1094, 1261–1395). The two well-structured domains, TPR and JmjC,
are connected by an intrinsically disordered region (IDR) [[49]15].
Interestingly, a high number of other chromatin-associated proteins,
such as Nucleophosmin, p300 and KMT2A-D, possess such flexible
sequences, which seem to be common and crucial motifs for interactions
with proteins and DNA/RNA [[50]16].
In cancer, KDM6A mutations can be found throughout the whole coding
region, most prevalently within the functionally relevant domains.
Knowing the precise effect of mutations on the functionality of KDM6A
is key to fully understand the pathogenic effects resulting from
impairments of this protein and to develop strategies for targeted
therapies. Therefore, we developed a workflow to systematically and
simultaneously follow changes of the intrinsic properties of KDM6A
substitution and truncation variants, using eGFP as an all-in-one
fluorescence marker and affinity tag. We characterized the impact of
KDM6A mutations on (i) demethylase activity by an ELISA-based
demethylase assay, (ii) protein stability by western blotting, (iii)
intracellular (co-)localization by immunofluorescence, scatterplot
analysis and cellular back-mapping, (iv) complex assemblies by
co-immunoprecipitation (Co-IP) and (v) cell death using an Annexin V
based flow cytometry analysis.
Results
Generation of amino acid substitution and truncated variants of KDM6A based
on the mutational landscape of KDM6A in tumor tissues and cell lines
Based on 64,668 tested samples from 44 tissue types, 2496 unique
mutations are listed for KDM6A in COSMIC v92 (GRCh 38, November 2020).
Among the cancer tissues, meninges and the urinary tract exhibit the
highest mutation frequency in each more than 30% of the tested cancer
samples (411/1336 cases for UC). The three most frequent point
mutations across all tissues are the synonymous mutation
Q1037 = (c.3111G > A) in the JmjC domain, the missense mutation T726K
(c.2177C > A) and the truncating Q555* (c.1663C > T), both in the
intrinsically disordered region. We selected T726K as a hotspot
substitution variant as well as substitution mutations typically found
in urothelial cancer cell lines and tissues: E315Q (located in TPR6)
and D336G (TPR7), P966R (loop in a beta sheet cavity containing the
active center), V1338F (JmjC, zinc-binding) and C1361Y (JmjC,
zinc-binding). To compare the impact of these substitution mutations,
we additionally selected the variants Q1133A (H3-tail interaction,
JmjC) and H1329A (hydrophobic patch near active center, JmjC) known to
be catalytically inactive [[51]14]. Positions P966, Q1133, V1338 and
C1361 are conserved in the KDM6A zebrafish ortholog and the closest
functional KDM6A paralogs KDM6B and KDM6C. We created all substitution
variants by site-directed mutagenesis using eGFP-KDM6A as a template
(Fig. [52]1A). As nonsense mutations make up nearly one quarter of all
point mutations and generate truncated variants with partial losses of
the IDR and JmjC, we established a set of truncated variants, each with
an eGFP-tag fused to the N-terminus: ΔTPR, ΔIDR, ΔJmjC, TPR (=
ΔIDR/ΔJmjC) and JmjC (= ΔTPR/ΔIDR) (Fig. [53]1C). These variants were
transiently transfected into urothelial cancer cell lines with wildtype
(SW-1710) or mutated (T-24) KDM6A/KMT2C/D proteins to study the
influence of the endogenous proteins on the cellular response after
reintroduction. All KDM6A substitution and deletion variant proteins
were detectable at the expected sizes (Fig. [54]1). In a second step,
we analysed all variants for their demethylation activity. KDM6A JmjC
and the flanking zinc-binding domain are known to recognize and bind
several amino acids between H3R17-H3T32 of the H3K27 di- and
tri-methylated N-terminal histone tail to ensure substrate specificity
[[55]14]. Consequently, mutations in the JmjC and flanking domains have
a high potential to impede or even abolish the catalytic activity.
However, it is unknown to what extent mutations in the TPR and IDR
might contribute to KDM6A demethylase activity.
Fig. 1.
[56]Fig. 1
[57]Open in a new tab
Protein expression and activity of KDM6A substitution and deletion
variants. A Results of the ELISA-based activity assay of KDM6A WT and
substitution variants are displayed as normalized absorption on the
y-axis, which positively correlates with demethylated product.
Calculated amount of protein is depicted on the x-axis in logarithmic
scale. WT (black) and substitutions variants in TPR (blue) and IDR
(grey) generate more demethylated product with rising input amounts.
Substitutions affecting the JmjC domain (yellow) remain mostly at
baseline levels. T726K activity (#4A) increases with input amount at
24 h (grey boxes) but not at 48 h (grey stars). Error bars represent
the standard deviation between the triplicates of each individual
experiment (n = 4–18, depending on variant) C Graphic display of KDM6A
truncation variants. B/D Western blot showing all substitution and
deletion variants 48 h after transient transfection, detected with
a-eGFP (CST) and a-KDM6A (CST) antibodies, as well as WDR5 (CST) as
lysate control. Variants with IDR deletions are not detectable with the
a-KDM6A antibody, as it recognizes epitopes within the IDR. The ΔIDR
variant was enriched via IP. Color code blue-grey-yellow according to
the site of mutation (as depicted in A and C)
KDM6A demethylase activity is strongly affected by substitutions and
deletions within the JmjC domain
To assess the demethylase activity of KDM6A variants, we established an
ELISA-based H3K27me3 demethylation assay to screen for the catalytic
activity of all truncated and substitution variants. The assay was
carried out with the purified protein attached to GFP-dynabeads. We
used fluorescence emission of the eGFP to determine and normalize the
amount of protein before and after binding to dynabeads (Figure
S[58]2A-C). The activity of the variants was measured by colorimetric
readout of the demethylated products and subsequent fitting via
4PL-regression. The eGFP-KDM6A WT species served as a reference point
for demethylase activity (Figure S[59]2D-G). The activity of
substitution variants was assessed in a quantitative manner, yielding
the specific activities summarized in Table [60]1. As shown in Fig.
[61]1A, eGFP-KDM6A variant T726V featured a demethylase activity in the
range of WT. Note that TPR substitutions E315Q and D336G had a slightly
higher activity. Interestingly, activity in T726K decreased
time-dependently within 2 days after transfection (Figure S[62]2I).
H1329A and V1338F had strongly reduced activity, while P966R, Q1133A
and C1361Y were considered as non-active.
Table 1.
Specific activity of substitution variants
No. KDM6A variant, affected domain Specific activity
[10^− 3 μmol min^− 1 mg^− 1]
1 WT 2.15 ± 0.24
2 E315Q, TPR 3.96 ± 0.58
3 D336G, TPR 3.76 ± 0.89
4 T726K, IDR 0.69 ± 0.10
5 T726V, IDR 1.54 ± 0.11
6 P966R, JmjC < 0.07 ± 0.03
7 Q1133A, JmjC < 0.09 ± 0.02
8 H1329A, JmjC 0.28 ± 0.05
9 V1338F, JmjC 0.29 ± 0.03
10 C1361Y, JmjC < 0.12 ± 0.03
[63]Open in a new tab
Specific activity of KDM6A variants depends strongly on the mutation
site. Mutations affecting the JmjC domain impair catalytic activity,
whereas TPR substitution mutation enhanced activity. The IDR mutation
T726K showed time-dependent diminution of activity. Specific activity
was calculated as described in Figure S[64]2
The demethylase activity of the truncated KDM6A variants was assessed
in a qualitative manner since changes in the fluorescence emission
spectra of the truncated variants did not allow proper quantification
of the protein amount used in the assay (Figure S[65]2C). As expected,
experiments with truncated (Δ- and ΔΔ-) variants yielded activity only
when the JmjC domain was preserved (Figure S[66]2H).
KDM6A single substitutions do not alter nucleoplasmic localization
In SW-1710 and T-24 cells eGFP-KDM6A WT was located in the nucleoplasm
and in the cytoplasm, sometimes with a tendency to cytoplasmic speckle
formation (dependent on the dose of the transient overexpression). All
substitution variants showed cytoplasmic and predominantly
nucleoplasmic localization with different degrees of cytoplasmic
speckle formation, as well as weak accumulation around the nucleoli
(Fig. [67]2A and Figure S[68]4A). To rule out unspecific or GFP driven
localization of transfected KDM6A, we analyzed free eGFP controls in
SW-1710, T-24 and HBLAK cells (Fig. [69]2A, Figures S[70]3/[71]4). Free
eGFP as well as eGFP-KDM6A shows both cytoplasmic and nuclear
localization. The main difference between eGFP and eGFP-KDM6A is the
additional presence of free eGFP in nucleoli while eGFP-KDM6A is
exclusively found nucleoplasmic. The localization of eGFP-KDM6A is
similar to that of the endogenous KDM6A in these cell lines by
immunostaining using two different KDM6A antibodies (sc-514,859 and
CST-3351, Figure S[72]3).
Fig. 2.
[73]Fig. 2
[74]Open in a new tab
Localization of substitution and deletion variants. A Localization of
transiently transfected KDM6A substitution variants in SW-1710 (for
T-24 see Figure S[75]4) using confocal microscopy and post-processing
with HuygensPro 20.08. All variants localized to the nucleoplasm and
cytoplasm. Occasionally, KDM6A positive micronuclei and cytoplasmic DNA
were observed for all variants including wildtype (arrows). B Abnormal
localization pattern compared to the eGFP-KDM6A WT, WT[cloning] and
substitution variants: Leakage of DNA from the nucleus and subsequent
extranuclear DNA patches are common features with all truncated
variants. Scale bar is 20 µm
KDM6A transport into the nucleus depends largely on the KMT2C/D COMPASS
complex [[76]17]. Therefore, the observation that KDM6A WT and all
substitution variants localized in the nucleus suggests functional
nuclear import, as well as interaction with the COMPASS complex.
Notably, a recent study [[77]17] found that KDM6A TPR mutations, among
them the D336G variant, predominantly localized in the cytoplasm in
stably transfected HeLa cells. We performed a pull-down experiment of
KDM6A WT, D336G and T726K variants with RBBP5 to test for impaired
association with the KMT2C/D complex. RBBP5 is a core component of KMT2
complexes that directly interacts with ASH2L, WDR5 and DPY30 within the
WRAD complex to bind specific chromatin sequences, stabilize and
activate the methyltransferase activity of KMT2 proteins. RBBP5 was
pulled down with all three KDM6A variants to similar extents, but not
with the eGFP control in both urothelial cancer cell lines
(Fig. [78]3), suggesting that they are present in the same complex.
Furthermore, perinuclear KDM6A speckles also stained DAPI-positive,
indicated newly formed micronuclei. Micronucleus formation, an
indicator of genomic instability, is however common in urothelial
cancer cell lines. An increased tendency for DNA release was identified
with some KDM6A substitution variants, which colocalized at cytoplasmic
DNA sequences (Fig. [79]2, catalytically impaired variants). This
phenomenon was much stronger in KDM6A truncation variants.
Fig. 3.
[80]Fig. 3
[81]Open in a new tab
RBBP5 and NPM1 interaction analysis. Top section: KDM6A variants used
in the interaction study. Mid/Bottom section: IP of KDM6A variants with
RBBP5 and NMP1 in SW-1710 and T-24 (n = 2–7). The cleared cell lysates
(left section) indicates GFP, RBBP5 and NMP1 protein levels after
transfection of KDM6A WT, variants and the eGFP control. For SW-1710
(right section, mid) and T-24 (bottom), IP was used to pull RBBP5 with
eGFP-KDM6A WT, D336G, T726K, ΔTPR, ΔJmjC, TPR, JmjC and the control
eGFP. In SW-1710, NPM1 is slightly enriched in the IP in the eGFP-KDM6A
ΔTPR and ΔJmjC variants (white stars), whereas in T-24, NPM1 is
enriched in eGFP-KDM6A T726K variant (n = 1). The TPR and RBBP5 signals
are separately shown in Figure S[82]5, as well as controls and raw
Western Blot images
KDM6A truncated variants cause severe nuclear damage
Compared to the eGFP and eGFP-KDM6A WT controls, deletion of any
functional domain resulted in a heterogenous cellular response with
respect to subcellular distribution, localization and nuclear
integrity. KDM6A variants displayed a weak cytoplasmic and
nucleoplasmic localization and in some varinats presented a speckled,
perinuclear distribution of the transfected protein associated with
nuclear defects shown in Figs. [83]2 and S[84]4. Cellular responses in
both cell lines were comparable. For comparision, we transfected the
KDM6A TPR and JmjC variants into HBLAK cells, a non-transformed
urothelial cell line, and observed similar damaging effects. Nuclear
DNA, which was excessively released into the cytoplasm, colocalized
with KDM6A truncation variants. These results indicate the intrinsic
ability of all variants to bind chromatin, either directly through the
JmjC or indirectly via other protein-protein interactions by
TPR-containing variants. The presence or absence of the intrinsically
disordered region (IDR) strongly affects stability and solubility of
KDM6A. All variants principally localized to the nucleus, but (to
various extents) showed anomalies like partial nuclear redistribution
to nucleoli or accumulation in perinuclear DNA-associated speckles.
These observations raised two further questions, namely (1) how
deletions of one or two KDM6A domains impair functional interactions
with known interacting proteins such as RBBP5, the KMT2C/D (COMPASS)
complex and (2) whether Nucleophosmin (NPM1), a prominent shuttling and
chaperone protein found especially in nucleoli, may be involved in
KDM6A interactions with these organelles. To address these questions,
we performed protein immunoprecipitation (Co-IP) and co-staining of
transiently transfected KDM6A variants with NPM1 and RBBP5.
Full-length KDM6A is needed for maximal binding of the COMPASS-complex core
component RBBP5
First, we tested for RBBP5 interaction with KDM6A by Co-IP experiments
48 h post transfection of selected KDM6A truncation variants in T-24
and SW-1710 (Fig. [85]3). We had previously shown that RBBP5 was
enriched in KDM6A-tagGFP2 Co-IPs in different urothelial cancer cell
lines [[86]18]. Interestingly, we observed that for maximum interaction
with RBBP5 all KDM6A domains are needed (Fig. [87]3). Deletion of any
domain impaired RBBP5 binding. Specifically, TPR-containing variants
(ΔJmjC and TPR, Figure S[88]5) and to some extent IDR-containing
variants (ΔTPR) bound RBBP5 to some degree, but the JmjC domain alone
did not at all.
Intriguingly, proteomics analysis from two KDM6A-tagGFP2 stably
transduced urothelial cancer cell lines showed a significant enrichment
of nucleolar proteins, such as Nucleolin, NPM1 and ribosomal subunits,
in addition to histone variants (Figure S[89]6). This enrichment was
not seen with eGFP alone. However, in Co-IP experiments NPM1 could only
be significantly enriched with the KDM6A T726K mutant in T-24 (Fig.
[90]3), whereas other selected KDM6A variants failed to interact or
gave very weak bands (ΔTPR and ΔJmjC, Fig. [91]3 white stars).
Therefore, we searched for a complementary approach to detect
associations of KDM6A with NPM1.
Scatterplots are widely used to detect colocalization. Typically, the
intensities of two channels are plotted in a xy-diagram and the
resulting populations elucidate on colocalization, which is displayed
in Pearson (P between − 1 and 1) or channel-wise Manders values (M1, M2
between 0 and 1). However, one drawback of such values is that the
connection between scatterplot populations and the spatial cellular
information is lost. For this reason, we used images from a single
z-slice and generated scatterplots to identify populations of interest,
mapped them back to the respective cellular compartment using the
freely available Fiji plugin ScatterJ [[92]19] and compared the outcome
with line profiles through the corresponding cellular compartments. By
this approach, we identified localization changes among the KDM6A
variants in SW-1710 cells.
KDM6A WT and NPM1 form nucleoplasmic populations
As shown in the scatterplot for KDM6A WT and NPM1 (Fig. [93]4A, left
panel), two unique fractions appeared at the y- or x-axis, representing
signals in only one of the two channels. The corresponding eGFP-KDM6A
WT-only fraction is shown in green and the NPM1-only fraction in red. A
third fraction with green and red signals of different intensities is
shown in orange and named from here on “intermediate” fraction from
here on. Cellular back mapping of the selected populations
(Fig. [94]4A, middle panel), clearly showed the KDM6A WT-only signal
predominantly in the cytoplasm and only a minor fraction in the
nucleoplasm. As expected, the NPM1-only signal was present in nucleoli
and in nucleoplasm. The intermediate fraction was always associated
with the NPM1-only fraction at the nucleoli rims and within the
nucleoplasm, indicating a dynamic exchange of NPM1-only, mixed
complexes and KDM6A WT-only fractions at specific sites within the
nucleus. The line profile through the nucleus with DAPI as a DNA
indicator (Fig. [95]4A, right panel) corroborates these findings: High
NPM1 (red) signal intensities were exclusively found in nucleoli,
whereas green-red overlapping signals might represent KDM6A WT-NPM1
assemblies.
Fig. 4.
[96]Fig. 4
[97]Open in a new tab
Cellular back mapping identifies co-localizing NPM1-KDM6A protein
populations in nucleoplasm and at extranuclear DNA. A-C Left panel:
Scatterplot presentation of KDM6A (green box) and NPM1 (red box) and
one intermediate population representing a KDM6A-NPM1 assemblies
(orange box). Middle panel: All three populations were mapped back to
the cellular compartment. Right panel: Line profile through the nucleus
as indicated by an arrow in the image. DAPI (blue), NPM1 (red) and
KDM6A variants (green) signals are overlaid. The orange triangles
indicate the intermediate populations from the scatterplot. A Analysis
of KDM6A WT and NPM1. B Analysis of KDM6A T726K and NPM1. C Analysis of
KDM6A JmjC variant with NPM1 in the nucleus and at extranuclear DNA.
Scatterplot and back mapping analysis for KDM6A variants and DAPI is
shown in Figure S[98]7
The T726K hotspot mutant is absent from nucleolamina and additionally forms
cytoplasmic complexes
By comparing the scatterplot presentations from T726K or WT KDM6A and
NPM1, we observed a much broader intermediate fraction with similar
KDM6A-T726K signal intensities but higher NPM1 intensities
(Fig. [99]4B), which was also visible in the line profile. By cellular
back mapping, we identified the intermediate population in the
nucleoplasm and in the cytoplasm, where KDM6A T726K (green population)
was also detectable. In contrast, the nucleoplasmic NPM1-only fraction
was completely absent with this mutant. In the original image (Figure
S[100]7A), DAPI staining clearly indicated DNA release into the
cytoplasm. Scatterplot analysis of KDM6A T726K with DAPI (Figure
S[101]7B) showed KDM6A T726K associated to DNA in the nucleus and at
the released DNA (population 3). Furthermore, we identified a DAPI-only
fraction (population 4), which was also negative for NPM1 at the
nuclear lamina.
Truncated KDM6A variants likewise form complexes with NPM1 at extranuclear
DNA segments
As described above, truncated KDM6A variants elicited more severe
nuclear DNA release and nuclear damage (Fig. [102]2). As an example, we
selected KDM6A JmjC and KDM6A TPR variants (Fig. [103]4C) for further
analysis. Scatterplot analysis and line profiling of the truncated
variants with NPM1 clearly indicated an enriched, colocalizing
intermediate fraction at extranuclear DNA segments. As observed before,
KDM6A JmjC and TPR variants were also localized to the cytoplasm. In
contrast to KDM6A WT, these variants were always associated with DNA,
as shown by DAPI-KDM6A scatterplot analysis and cellular back mapping
approaches (Figure S[104]7B/C). By combining these approaches, we
identified KDM6A-NPM1 assemblies, which were hardly detectable by
Co-IP/WB analysis (Fig. [105]3), which could be caused by experimental
characteristics (proportion of extranuclear DNA segments is too little
compared to whole cell lysate) and resolution limits of the respective
experiment (here fluorescene imaging is superior to Co-IP).
Importantly, scatterplot analysis and the cellular back mapping
approach highlighted differences among the wildtype, substitution and
truncated KDM6A variants. The T726K variant represents a non-wildtypic
phenotype with reduced enzymatic activity in a time-dependent manner
and sometimes even DNA release. The severe phenotype of the truncated
variants is characterized by a massive DNA release and forms nearly
equal complexes with NPM1.
The severe phenotype of KDM6A truncation variants is characterized by mitotic
defects, DNA release and significantly decreased cell viability
As DNA release has been observed, we analysed whether the typical
indicators for a DNA damage response are activated after introduction
of KDM6A truncation variants, namely accumulation of phospho-γH2A.X and
RAD51. Especially JmjC containing variants colocalize with both markers
at chromatin bridges (Fig. [106]5C). Furthermore, we detected elevated
levels of phospho-γH2A.X with truncated KDM6A variants, whereas overall
phospho-γH2A.X levels were unchanged in KDM6A WT transfected cells,
even in cells with strongly overexpressed KDM6A WT protein (Figure
S[107]8). Visual inspection of phospho-γH2A.X in cells with truncated
variants revealed defects in mitosis (Fig. [108]5). These defects
occurred in anaphase as lagging chromosomes, multiple fragmentation
events, in telophase and cytokinesis by persisting chromosome bridges
and accumulation of DNA damage sites at chromosome bridges. To quantify
our observations, we established a scoring system to determine the
amount of cells with severe phenotypes per variant (Fig. [109]5A).
Here, we discriminated between mono- and binucleated cells and
quantified cells with damages (cytoplasmic DNA release, extreme nuclear
deformation, lagging chromosomes and chromosome bridges), micronuclei,
normal interphase or mitosis. Both KDM6A deletion variants, TPR and
JmjC, elicited a significant increase in DNA damage in mono- and
binucleated cells (Fig. [110]5B). We observed a trend for cells
transfected with eGFP-KDM6A WT, TPR and JmjC to be detected in
binuclear cells. To evaluate whether the nuclear damage promotes
apoptosis, we performed an Annexin V-based apoptosis assay 48 h post
transfection. The FACS measurements indicate that cell viability
significantly decreases in eGFP-KDM6A TPR and JmjC variants in both
urothelial cancer lines (Fig. [111]6A/B). This mirrors the results of
the scoring experiments very well, pointing towards a direct connection
between the nuclear damage and apoptosis induction.
Fig. 5.
[112]Fig. 5
[113]Open in a new tab
Truncated KDM6A variants promote a significant decrease in cells with
normal phenotypes in T-24 and SW-1710 cells. A Scoring criteria used to
quantifiy the occurrence of nuclear damage. B Frequency of different
phenotypes scored as normal interphase (grey) and mitosis (pink),
micronuclei (blue) and damage (red) in bi- or mononuclear T-24 and
SW-1710 untransfected (UT, 1) cells or transfected with control
eGFP (2), eGFP-KDM6A variants WT (3), TPR (4) and JmjC (5) (for
simplicity abbreviated as WT, TPR and JmjC). In mononuclear and
binuclear cells, the proportion of normal cells decreased significantly
for both the eGFP-KDM6A TPR and JmjC compared to eGFP and eGFP-KDM6A
WT. Cells with micronuclei were not significantly enriched. This was
largely due to an increase in damaged cells. Overall, we observed a
non-signifcant trend towards an increased number of binucleated cells.
T-test using two-tailed hypothesis, significance levels: * = P ≤ .05,
** = P ≤ 0.01, *** = P ≤ 0.001, see Table S[114]3 for P-values. C Line
profiles through lagging chromosomes and the “knot-like structures” of
the chromatin bridges found in samples transfected with eGFP-KDM6A
TPR (left) or JmjC (right) indicating overlapping signal intensities of
KDM6A variants (green) with DNA damage markers RAD51 and p-γH2AX (red)
Fig. 6.
[115]Fig. 6
[116]Open in a new tab
Truncated KDM6A variants decrease the amount of viable in T-24 and
SW-1710 cells and exhibit mitosis errors. A FACS analysis in Annexin V
based apoptosis assay. Transfected eGFP positive cells were gated based
on the threshold obtained from untransfected cells (UT), which were
then used to plot PI (for membrane permeability) against Annexin-V-APC
(apoptosis marker). The plot was divided into four quadrants,
representing the viable population (lower left, grey), early apoptosis
(lower right, green), late apoptosis (upper right, dark cyan) and
necrosis (upper left, black). B Statistics derived from triplicate
measurements. eGFP-KDM6A TPR and JmjC show a significant decrease in
cell viability in comparision to the eGFP control in both cell lines.
T-test using two-tailed hypothesis, significance levels: * = P ≤ .05,
** = P ≤ 0.01, *** = P ≤ 0.001, see Table S[117]3 for detailed
P-values. C Graphic summary of cellular phenotypes observed with KDM6A
mutation variants, depicting the impact on localization, mitosis,
apoptosis and protein assemblies
Discussion
In this study, we developed and utilized a comprehensive analysis tool
kit to understand the relationship and interplay of known and predicted
regulatory features of the multi-domain protein KDM6A. We demonstrate
how single substitution mutations and deletions of the main three
functional and regulatory domains, TPR, IDR and JmjC, affect the
intrinsic properties of the target protein and its interactions with
the cellular environment. We could show that mutations within the JmjC
domain affected the catalytic activity. Although nuclear localization
changed little, interactions with RBBP5 and NPM1 were affected,
especially in truncated variants, where we observed a cellular
mechanism to dispose of harmful KDM6A variants, namely by
cytoplasmically released DNA-KDM6A complexes. Harmful KDM6A variants
cause mitosis defects and DNA damage which promotes cell death.
First, we developed an ELISA-based demethylase assay suited for use
with eGFP-tagged KDM6A variants. Our experiments revealed, that
substitution variants within the JmjC (catalytic domain) possessed
reduced or abolished demethylase activity, especially if amino acids
involved in peptide recognition, peptide binding or stabilizing were
changed. Remarkably, the demethylase activity was also reduced in the
IDR-variant T726K, but not in T726V. Therefore, we predict a unique
functionality for the amino acid position K726 (Table [118]1, Figure
S[119]2). Among the selected cancer-associated point mutations, the
hotspot mutation T726K is the third most frequently listed one in
Cosmic v92 across all tissues and was the only substitution variant
yielding a time-dependently lowered demethylase activity. We predicted
and tested K726 as a possible methylation site, but mass spectrometry
analysis did not show any PTM at K726. The demethylase activity of
truncated KDM6A variants was determined by the presence or absence of
the JmjC domain. As expected, KDM6A JmjC, ΔTPR and ΔIDR exhibited
demethylase activity, whereas TPR and ΔJmjC did not (Figure S[120]2).
However, removal of other domains negatively affected protein stability
and solubility, especially for KDM6A ΔIDR (Table S[121]1). While those
variants with N-terminal or central truncations are mostly artificial,
C-terminally truncated variants generated by nonsense mutations make up
almost a quarter of all listed KDM6A mutations in COSMIC v92. A
prevalent nonsense mutation is Q555*. Its prevalence may be partly
explained by the observation that it represents a hotspot for
APOBEC3-mediated mutations [[122]20], which are frequent in multiple
cancer types including bladder cancer [[123]21]. Functionally, the
Q555* variant fully lacks the JmjC domain and has a partial deletion of
the IDR. Such IDR/JmjC nonsense mutations would abolish demethylase
activity by truncation or deletion of JmjC. Another example is the
KDM6A variant found in the urothelial cancer cell line T-24 with
heterozygous mutations at E895* and E902*. These similar-sized variants
are still expressed endogenously and can be detected as a ~ 97 kDa band
on WB [[124]18]. It remains speculative whether these fragments display
dominant negative effects and are actively involved in generating the
cancerous T-24 phenotype. Other variants, like the moderately frequent
Q333*, lack both IDR and JmjC and could potentially impair the TPR8.
Apart from lost demethylase activity, these variants likely have
impaired interactions with other proteins. We have recently shown that
KDM6A associates with RBBP5 in urothelial cancer cell lines dependent
on the mutation status of KDM6A and KMT2C/D proteins [[125]18]
suggesting vital interactions of KDM6A with the COMPASS complex. Here
we showed that TPR substitution variants did not affect RBBP5 binding.
However, especially the KDM6A mutation D336G has been shown to be
predominantly cytoplasmic in HeLa cells presumably due to impaired
binding to ASH2L in a pull-down experiment and reduced nuclear import
by the KMT2C/D complex [[126]17]. In a previous study we observed that
KDM6A nuclear import was strongly decreased after double, but not
single, knock down of KMT2C and KMT2D proteins [[127]18]. Systematic
deletion of KDM6A domains clearly indicated that all domains, including
TPR and IDR, are necessary for binding of RBBP5 (Fig. [128]3)
independent of demethylase activity. Although JmjC alone does not bind
RBBP5, the presence of this domain enhanced binding in KDM6A WT
compared to KDM6A ΔJmjC and KDM6A TPR. A recently published study
indicated that RBBP5, WDR5 and the KDM6A JmjC domain share similar
recognition and binding motifs at the Histone 3 tail (aa 1–57)
[[129]20]. Furthermore, RBBP5, but not WDR5, bound to H3K27me3 modified
peptides and K27F.
All KDM6A variants were located in the nucleoplasm, albeit to different
extents, independent of their mutation status. Truncation variants, but
not substitution variants, are characterized by eliciting (1)
cytoplasmic DNA release, (2) enhanced levels of RAD51 and phospho-γH2AX
as indicators of DNA damage, and (3) defects of mitosis caused by
missegregated chromosomes at anaphase and persisting chromatin bridges
at telophase and cytokinesis (Figs. [130]2, [131]5, S[132]4). All
observed effects occurred on a short time scale within 36–48 h. While
transient and stable overexpression of the KDM6A WT reduces long-term
cell growth and colony formation [[133]18], we never observed effects
of this kind, neither short-term nor long-term. In general, aneuploidy,
replication stress and mitosis errors are common in cancers [[134]21].
Accordingly, all cancer cell lines used in this study exhibit these
features, but they are profoundly enhanced after induction of KDM6A
truncation variants (Fig. [135]5). Among the severe phenotypes,
cytoplasmic DNA release was most commonly observed. All KDM6A
truncation variants were associated (directly or indirectly) with the
DNA released from the nucleus as indicated by localization analysis and
the cellular back mapping approach. Nuclear DNA release is the presence
of cytoplasmic DNA caused by a yet unknown mechanism. We speculate that
appearance of cytoplasmic DNA could be caused by (1) pulverized
micronuclei or (2) chromosome fragments without envelope or (3) active
nuclear release due to impaired nuclear integrity or DNA damage
[[136]22]. Our observations point towards mitotic defects (Fig. [137]5)
followed by apoptosis (Fig. [138]6). However, we cannot rule out
additional mechanisms, as we do not have conclusive data on cGAS/STING
activation that is expected in response to cytoplasmic DNA accumulation
[[139]23]. Moreover, introduction of KDM6A variants, especially
truncated ones, elicited elevated phospho-γH2A.X levels (Figure
S[140]8). Phospho-γH2A.X is activated during the DNA damage stress
response [[141]24]. Enrichment of proteins involved in DNA repair and
stress response (DDR) appeared in our MS-data analysis from three
different urothelial cancer cell lines with stably or transiently
transfected KDM6A WT (Figure S[142]6). Notably, KDM6A activity in
differentiating embryonal stem cells has been linked to DNA damage
response pathways by colocalization with γH2A.X positive foci
[[143]24]. In addition, as an oxygen-dependent enzyme KDM6A serves as a
sensor to control chromatin and cell fate [[144]25]. Thus,
overexpressed (with a high dose-effect) and impaired KDM6A variants as
well as oxygen-related stress have the tendency to increase DNA damage.
This phenomenon was also observed in diabetic kidney disease [[145]26].
An additionally prominent feature of truncated KDM6A variants was a
high degree of co-localization with NPM1 at extranuclear DNA and they
appeared, like all variants, as a mixed population in the nucleoplasm
(Fig. [146]4) as suggested by scatterplot and cellular back mapping
analysis. As NPM1 is involved in rRNA processing, ribosome maturation
and shuttling of ribosomal subunits between nucleoli, nucleoplasm and
cytoplasm [[147]27], KDM6A might be involved in these processes, too.
However, the co-occurrence of NPM1 and KDM6A truncation variants may
rather result from the role of NPM1 as a chaperone [[148]28]. At this
stage, we cannot completely rule out activation of the unfolded protein
response pathway (UPR) or ER proteostasis [[149]29] by truncated KDM6A,
but consider it rather unlikely for the following reasons. (1) We
observed correct nuclear localization of all truncated variants (Figure
S[150]4). (2) Perinuclear aggregation was observable in all KDM6A WT,
substitution, truncation and control (eGFP) variants. (3) In truncated
variants with a severe phenotype, KDM6A protein was always associated
with DNA and never freely distributed throughout the cytoplasm. It
remains possible that UPR stress sensors contribute to activation of
the nuclear DNA damage response [[151]29], which is confirmed by
enhanced phospho-γH2A.X levels. As KDM6A itself might act as a critical
stress sensor, it is difficult to ascertain at this stage which
signaling cascade might explain our observations best.
Our observations hint at possible new functions or involvement of KDM6A
in the cell cycle. Specifically, the following questions are raised:
First, what is the role of KDM6A during mitosis and to which extent is
any such function dependent on its catalytic activity and its interplay
with RBBP5 and further components of the KMT2C/D-COMPASS complex?
Notably, many lysine demethylases (KDM) have cell cycle specific roles
[[152]30, [153]31]. KDM4C, KDM1A and KDM7B have already been linked to
mitosis by regulation of chromosome segregation, transcriptional
activation of mitotic checkpoint complex components (see refs in (30)).
Moreover, WDR5 and KMT proteins, likely KDM6A interaction partners,
have also been shown to be involved in mitosis [[154]32, [155]33]: WDR5
is part of the midbody in the spindle apparatus [[156]31].
Intriguingly, we found endogenous KDM6A located along the midbody
(Figure S[157]8). Secondly, the multifaceted functions of NPM1 in
chromatin remodeling, DNA repair, cell cycle control, apoptosis,
mitotic spindle, centromere and cytoskeleton binding [[158]28, [159]34]
and its prominent enrichment in MS analysis suggests an important link
between both proteins. Thus, under which conditions and in which manner
do both proteins directly or indirectly interact?
Conclusion
Overall, our approach combining biochemical, cellular and imaging
techniques revealed that truncating KDM6A mutations lacking TPR, JmjC
and/or IDR dramatically increase nuclear damage and apoptosis whereas
single substitution variants with diminished demethylase activity or
unknown features (T726K) show at most a weak trend towards these
effects which are summarized in Fig. [160]6C. Our findings point
towards a pivotal balance between the different domains of KDM6A,
which, if disturbed, might facilitate interference of these KDM6A
variants with cellular processes mediated by the wildtype protein and
its partners.
Methods
Cell lines and cell culture
Parental T-24 and SW-1710 urothelial carcinoma cell lines were obtained
from the DSMZ (Braunschweig, Germany). Cells were cultured and treated
in DMEM GlutaMAX-I (Gibco, Darmstadt, Germany) with 10% fetal bovine
serum (FBS; Gibco™, Thermo Fisher Scientific) and 100 U/ml
penicillin/100 μg/ml streptomycin (Sigma-Aldrich, Darmstadt, Germany),
except for HBLAK cells, which were solely cultured in CnT-Prime
Epithelial Culture Medium (CELLnTEC, Bern, Switzerland) without any
additives. Cells were incubated at 37 °C in a humidified atmosphere
with 5% CO2. STR (short tandem repeat) profiling via DNA fingerprint
analysis was performed for all cell lines in this study and is
available upon request.
Generation of eGFP-KDM6A substitution and deletion variants
eGFP-KDM6A wildtype (WT) was synthesized by BioCat (Heidelberg,
Germany) by cloning a codon-optimized eGFP-KDM6A (both sequences
full-length, KDM6A main isoform 1, Uniprot ID [161]O15550, without
additional linker between eGFP and KDM6A) into the pcDNA 3.1(+) vector,
using NheI and NotI as flanking restriction sites. Generation of
eGFP-KDM6A substitution variants was done by using site-directed
mutagenesis (SDM) with 10 ng eGFP-KDM6A WT plasmid and mutagenesis
primers (Table S[162]2A). 1.25 U PrimeSTAR GXL DNA Polymerase (Takara
Bio, Kusatsu, Shiga, Japan) was used in PCR reactions. Successful PCR
amplification and product length was checked by gel electrophoresis.
After DpnI (NEB, Ipswich, MA, USA) digestion (20 U for 1 h at 37 °C),
SDM amplicons were transformed into Escherichia Coli XL10-Gold®
(Stratagene, Santa Clara, CA, USA) and spread on LB amp plates.
Colonies were picked, grown and DNA was isolated using a QIAprep Spin
Miniprep Kit (QIAGEN, Hilden, Germany). After sequencing, DNA from
positive KDM6A substitution clones was re-transformed into E. coli
XL10-Gold® (Stratagene) and purified at a large scale using NucleoBond
Xtra Maxi Plus EF kit (Macherey-Nagel, Dueren, Germany). Substitutions
were then re-confirmed by sequencing. Generation of eGFP-KDM6A deletion
variants was done by “modularized” cloning of three inserts: TPR, res.
1–390, IDR, res. 391–885 and JmjC, res. 886–1401 from the original
wildtype eGFP-KDM6A pcDNA3.1(+) were cloned into the pEGFP-C1 vector
for the desired combinations. Each restriction enzyme (RE) site
produces a two amino acids long linker. Constructs with one insert
(eGFP-TPR, eGFP-IDR) have BspEI and HindIII as flanking RE sites.
Constructs with two inserts (eGFP-KDM6A ΔTPR, ΔIDR, ΔJmjC) have BspEI
and EcoRI as flanking RE sites and HindIII as middle RE site. The
control construct eGFP-KDM6A[cloning] has BspEI and KpnI as flanking RE
sites and HindIII and EcoRI as mid RE sites. Amplification primer
(Table S[163]2A) were designed according to the desired combination
with appropriate overhangs and synthesized by Eurofins Genomics
(Ebersberg, Germany). T4 DNA ligase (NEB) was used for insert ligation
(10 min at RT, 3.1 insert:vector ratio). NucleoSpin Gel and PCR
Clean-Up (Macherey-Nagel) was used to extract and clean up DNA. Cloning
products were transformed into Escherichia Coli XL10-Gold®
(Stratagene). An appropriate number of colonies were picked, grown and
the DNA was isolated using a QIAprep Spin Miniprep Kit (QIAGEN). After
sequencing, positively cloned DNA was re-transformed into E. coli
XL10-Gold® (Stratagene) and purified in a large scale using a
NucleoBond Xtra Maxi Plus EF kit (Macherey-Nagel).
Transient transfection
For transient transfection, cells were seeded into 6-well plates with
glass cover slips (for imaging) or without (for activity, western
blot). 24 h later, cells were transfected at ~ 90% confluence using
X-tremeGENE™ 9 or HP (Roche, Basel, Switzerland; application dependent
use) in a 2:1 ratio (v/w) of transfection reagent to DNA. Total DNA
transfected per well (9.6 cm^2) did not exceed 2 μg. Transfection was
carried out for 24–48 h for activity assay and Western Blot
applications and 36 h for localization analysis.
Cell death analysis by flow cytometry
One hundred fifty thousand cells (SW-1710) and two hundred thousand
cells (T-24) per well of a six well plate were seeded and reversely
transfected with XtremeGENE™ HP (Roche). After 16 h cells were split
into two wells. 48 h post transfection, supernatant and cells were
collected, centrifuged at 1000 rpm for 5 min, washed with ice-cold 1x
Annexin binding buffer (Serva, Heidelberg, Germany) and centrifuged
again. The pellet was resuspended in 75 μl 1x Annexin binding buffer
containing 4.5 μl Annexin V-APC (Serva) and 7.5 μl PI (1 mg/ml, Serva)
and incubated for 15 min in the dark at RT. The suspension was diluted
with 500 μl 1x Annexin binding buffer, centrifuged, washed and fixed
with 0.5% methanol-free formaldehyde for 20 min on ice. The reaction
was stopped with 500 μl 1x Annexin binding buffer. FACS measurements
and analysis was performed using the MACSQuant Analyzer X and
MACSQuantify Software (Miltenyi Biotec, Bergisch-Gladbach, Germany). In
total, 50,000 cells/experiment were analyzed in three independent
experiments.
Co-IP and Western blot analysis
An appropriate amount of cells were lysed by suspension in SDS-free
RIPA like buffer (RLB) consisting of 50 mM Tris-HCl (pH 7.5), 0.3%
CHAPS, 150 mM sodium chloride, 1 mM sodium vanadate (Na[2]VO[4]), 10 mM
sodium fluoride (NaF), 1 mM ethylene diaminetetraacetate (EDTA), 1 mM
ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′,-tetraacetate (EGTA),
2.5 mM tetrasodium pyrophosphate Na[4]O[7]P[2]. Dithiothreitol and 1x
HALT™ protease inhibitor cocktail (Sigma-Aldrich) were freshly added.
Lysis was followed by immediate freezing in liquid nitrogen, thawing on
ice for 30 min and repeated mixing by pipetting for 30 s each. The
lysate was either directly separated by SDS-PAGE on a 4–20% gradient
gel or used in the following Co-IP steps. For Co-IP, the GFP containing
lysate was incubated with GFP-trap dynabeads (Chromotek,
Planegg-Martinsried, Germany) for 1 h at 4 °C with constant mild
agitation to pull-down eGFP-KDM6A variants and complexed proteins.
Dynabeads were magnetically separated and washed three times with Co-IP
buffer, resuspended in SDS-PAGE loading buffer, boiled and separated on
a 4–20% gradient gel. The gel was transferred onto an methanol
activated PVDF-membrane. The membrane was blocked in TBS/0.1% Tween
(TBS-T) and 5% BSA for 1 h at RT and subsequently incubated with the
respective primary antibody (Table S[164]2B) in TBS-T, 1% BSA overnight
at 4 °C. The membrane was washed three times in TBS-T at RT. The
secondary antibody (Table S[165]2B) was applied for 1 h at RT in TBS-T,
1% BSA. The membrane was washed again three times and Clarity™ ECL
(Bio-Rad, Hercules, CA, USA) was used to develop the signal with the
ChemiDoc Imaging System (Bio-Rad, USA).
ELISA-based demethylase activity assay
Preparation and lysis of the cells was done the same way as described
for Co-IP. However, before the GFP-trap dynabeads were added, the
fluorescent emission signal of the lysate was measured (470 nm ex.,
485–650 nm em.) in a microliter cuvette. The dynabeads were then mixed
in the lysate for 1 h at 4 °C under constant, mild agitation. Beads
were then magnetically separated, the fluorescent emission of the
remaining lysate was measured again with the same specifications as
above. The delta in the emission spectra before and after bead
incubation in the range of 500–530 nm was used to calculate the amount
of eGFP-KDM6A pulled out of the lysate in each run. To eliminate
residual RLB buffer, beads were washed two times with the activity
assay (AA) buffer (50 mM TRIS-HCl (pH 7.45), 0.02% Triton X-100, 100 μM
α-ketoglutarate, 50 μM Fe(NH[4])[2](SO4)[2]*6 H[2]O, 100 μM ascorbic
acid, 1 mM TCEP, cofactors and TCEP being added freshly to avoid
oxidation. H3K27me3 (ProteoGenix, Schiltigheim, France) and H3K27me2
(BioCat) peptides (Table S[166]2C) were dissolved in AA buffer and
mixed with the loaded beads. The beads were incubated with the peptides
for 4 h at 30 °C while maintaining constant suspension. Afterwards, the
beads were magnetically separated and the supernatant containing the
biotin-labeled peptide was loaded onto a streptavidin-coated 96-well
plate (triplicate per variant/control, 50 μl per well). After 1 h of
biotin binding at RT and removal of the solution, the wells were loaded
with 50 μl of the a-H3K27me2 antibody in 0.1% TBS-T and incubated for
1 h at RT while shaking gently. The wells were then washed three times
with 150 μl TBS-T. Subsequently, 50 μl of a 1:1000 alkaline phosphatase
(ALP)-conjugated secondary antibody was added and incubated for 30 min
at RT. The wells were washed four times for 5 min. For detection,
100 μl of p-nitrophenyl phosphate (pNPP, Sigma-Aldrich) was added into
each well and incubated for 10 min at RT in the dark, mixing
thoroughly. The reaction was then quenched by 100 μl 1 M NaOH. The
signal was measured in a plate reader at 405 nm absorption, quantified,
normalized and fitted. To fit the ELISA readout, we used a
4-PL-regression normalized to the standard curve (Figure S[167]2). The
WT fit overlaid with the standard curve was used to directly calculate
the relation between the amount of fluorescence signal and demethylated
product. WT 4PL-regression fit was applied to calculate c[50] as a
reference point to compare the WT value with the variants.
Additionally, eGFP reference measurements were used to calculate the
absolute amount of protein input and calculate a specific activity.
Initial validation for the assay was done with recombinant full-length
KDM6A (Active Motif, Carlsbad, CA, USA).
Immunocytochemistry
Depending on the antibody requirements two protocols were used. For ICC
with primary antibody incubation overnight, cells were seeded on
coverslips, transiently transfected and fixed at > 80% confluence with
1% (v/v) para-formaldehyde, 0.02% (v/v) Triton X-100 for 20 min at RT.
Blocking and permeabilization was done with 1% (w/v) BSA, 0.1% (w/v)
saponin in PBS for 30 min at RT. After overnight night incubation at
4 °C, coverslips were washed with PBS and incubated with the secondary
antibody for 1 h at RT. Slides were washed with PBS, stained with DAPI,
washed again frequently with PBS and mounted. For ICC with primary
antibody incubation for 1 h RT, cells were prepared as before, but
fixed with 4% FA (v/v) for 10 min at RT. Permeabilization was done with
0.5% (v/v) Triton X-100 for 3 min at RT and blocking with 1% (w/v) BSA
in PBS for 30 min at RT. The primary antibody was incubated with
shaking for 1 h at RT, coverslips were washed with PBS and incubated
with shaking with the secondary antibody for 1 h at RT. Slides were
washed with PBS, stained with DAPI, washed again several times with PBS
and mounted. Primary and secondary antibodies are listed in Table
S[168]2.
Microscopy and image processing
Confocal imaging with live or fixed cells was performed on a confocal
laser scanning microscope FV1000 IX81 inverted microscope (Olympus,
Shinjuku, Japan) using a 60x water immersion UPLSAPO NA 1.2 objective.
DAPI, eGFP and Star Red were excited at 405 nm, 488 nm and 635 nm,
respectively, with the internal FV10-MARAD-2 main laser unit. Star
Orange was excited at 559 nm with an external Opti λ 559 diode laser
(NTT Electronics, Yokohama, Japan). Internal PMT detectors (Olympus)
were used for detection. Confocal laser scanning microscope (LSM)
processing routine was carried out with the freely accessible Fiji and
Huygens Pro 20.10 (SVI, Hilversum, Netherlands) for deconvolution. For
deconvolution of images fulfilling the Nyquist criterion, we used an
automatically computed theoretical point spread function based on our
known microscopic parameters and a model of the Olympus IX81 provided
by SVI and performed 30 iterative steps of classic maximum likelihood
estimation (CMLE) on our images. The signal-to-noise ratio was
determined for each channel and then kept constant during image
processing.
Scatterplot generation and cellular back mapping
We used the freely available, open source Fiji plugin ScatterJ
[[169]19]. Processed single slice images were converted into 8-bit grey
scale tagged image file formats (.tiff) and opened in ScatterJ.
256 × 256 pixel scatterplots were saved as xy-lists (.dat) for
processing in OriginPro. Within scatterplot, regions were selected
using Fiji free-hand-tool and back mapped to the original image. The
back-mapped image, as well as channel-wise images, were saved as text
sequences (.dat) or portable networks graphics (.png) for further image
and matrix analysis in OriginPro. The resulting cellular
back-mapping-images representing the pixel-wise analysis of defined
scatterplot populations are shown in Fig. [170]4 and the corresponding
Figure S[171]7.
Supplementary Information
[172]12860_2021_394_MOESM1_ESM.docx^ (18.7KB, docx)
Additional file 1: Table S1. Predicted solubility and pI of KDM6A
substitution and truncation variants. Predicted solubility and pI of
KDM6A substitution and truncation variants using Protein-Sol from the
University of Manchester based on published sequences [[173]35,
[174]36]. Compared to KDM6A WT protein TPR and ΔIDR show reduced
solubility, whereas the IDR sequence alone is predicted to be most
soluble. Single substitution do not affect pI and solubility, as
expected. Among nuclear proteins, such as components of the COMPASS
complex (KMT2C/D, WDR5, RBBP5) and NPM1, KDM6A is the least soluble.
Protein identifier were taken from UniProt.
[175]12860_2021_394_MOESM2_ESM.docx^ (15.3KB, docx)
Additional file 2: Table S2. Primer, antibodies and peptides used in
this study. A. Primer used for site-directed mutagenesis and
amplification. B. Primary and secondary antibodies used in this study.
C. H3K27-peptides used in this study.
[176]12860_2021_394_MOESM3_ESM.docx^ (14.6KB, docx)
Additional file 3: Table S3. P-Values and scoring results from
Figs. [177]5 and [178]6. A. P-Values from Fig. [179]5. B. Scoring
results used in Fig. [180]5. C. P-Values from Fig. [181]6.
[182]12860_2021_394_MOESM4_ESM.docx^ (784.3KB, docx)
Additional file 4: Figure S1. Bioinformatic analysis of KDM6A
tetratricopeptide repeats. A. Alignment of annotated KDM6A TPRs using
the TPRprediction and alignment tool published in [[183]37]. All
annotated KDM6A TPRs with the given amino acid range (see label) were
compared for their TPR conservation to the consensus sequence as
published in [[184]38] with the most conserved amino acids W4, L7, G8,
Y11, A20, Y24, A27 and P30. Identity and p-values are shown on the
right site. The TPR motif could not be identified for TPR3 (aa
170–199). Color coding according to CLUSTAL W alignment. B. Secondary
structure prediction of TPR3 using the Quick2D on the MPI
Bioinformatics toolkit server [[185]39, [186]40] indicated a high
probability for aa 14–26 to form an α-helix, but aa 1–10 tend to form
either an α-helix or a β-strand. Of note, the canonical aa 34 TPR
repeating unit forms a helix-turn-helix motif [[187]38]. In general,
the N-terminal TPRs 1–4 show less strict conservation towards the
canonical TPR sequence compared to the C-terminal TPRs 6–8.
[188]12860_2021_394_MOESM5_ESM.docx^ (345.2KB, docx)
Additional file 5: Figure S2. Activity assay and data analysis. In this
section, we describe our data analysis workflow. A. delta (grey shaded
area) in fluorescence emission spectrum of eGFP-KDM6A WT lysate before
incubation and after incubation with GFP-trap dynabeads. For data
analysis, the amount of pulled fluorescent protein was determined by
subtracting the eGFP fluorescence emission (470 nm excitation) after
incubation with GFP-trap dynabeads from eGFP fluorescence emission
before incubation with eGFP-trap dynabeads. Fluorescence intensities in
the range of 500–530 nm were integrated subsequently (later referred to
as integrated fluorescence). B. Maximum-normalized spectra obtained
from crude cell lysate of eGFP, eGFP-KDM6A WT or eGFP-KDM6A Q1133A
samples. All fluorescence emission spectra of the substitution variants
had a similar profile between 500 and 530 nm. This was used to compare
the amount of protein for each variant. Q1133A variant is shown
exemplarily for all other substitution variants. C. Fluorescence
emission spectra of WT and ΔJmjC. The high signal of ΔJmjC towards the
blue end of the spectrum is due to scatter, which could indicate
protein instability under the given (low salt) buffer conditions. D.
1–238 ng demethylated product (H3K27me2 peptide, MW = 2945.5 g/mol) was
obtained from at least nine data points from three independent
experiments, each in triplicates and fitted with a four parameters
logistic regression (4-PL) according to Eq. 1:
[MATH: y=d+a−d1+xcb :MATH]
(1) with y = assay readout absorption, x = amount of product in A.,
amount of KDM6A species, either by fluorescence in B. or amount of
protein in D., a = background signal, d = maximum signal, b = slope
factor, c = c50, x-value at half maximum y.The following fit results
were obtained: a = 0.29, b = 1.27, d = 1.73 and c = 62.4 ng. E. The
amount of protein was plotted against absorption (shown for KDM6A WT)
and fitted accordingly with 4-PL, with c being the only open parameter.
c50 for WT was determined with 518,000 +/− 56,000 AU. To correct for
background and maximum signal fluctuation between experiments, they
were normalized to the values obtained from the product curve fit
(a = 0.29, d = 1.73). F. A defined amount of recombinant eGFP (27 kDa)
was used to convert the x-axis from fluorescent signal to the amount of
protein. The linear dependency was used to calculate the amount of
eGFP-KDM6A (181 kDa) per fluorescent unit. The molar integrated
fluorescence (500–530 nm) from emission spectra for recombinant eGFP
(MW = 26.9 kDa) was determined as 2.34E+ 18 AU/mol from the relation 1
ng eGFP = 86,900 [AU]. Therefore, 1 ng eGFP-KDM6A WT would yield an
integrated fluorescence signal of 1.29E+ 03 AU. G. Data from E. and F.
were used to transform the x-Axis with the fluorescent signal into the
amount of KDM6A protein,. Approximated from D., the respective c50
always corresponded to 62.4 product. This correlation was used to
calculate the specific activity for each variant. Given the fixed time
of 240 min per assay, the specific activity under these conditions was
calculated. The c50 for all available substitution variants was
obtained in the same manner, fixing all parameters except c, using it
as a relative measure of activity. At least four independent triplicate
measurements were obtained for each substitution variant. H. absorption
for truncated variants, three repeats with triplicates each. Since the
truncated variants showed an unusual spectrum with a high scatter
fraction at lower wavelengths (see C.), a quantification was
impossible. Therefore, we qualitatively confirmed, that all truncated
variants with a JmjC domain are catalytically active (green boxes) and
those without JmjC domain are catalytically dead (red boxes). The
distribution of the signal over the background is an evidence for
activity in all truncated variants with a JmjC domain. ΔJmjC and TPR do
not exceed assay background levels (zero), while WT, ΔIDR, ΔTPR and
JmjC are all above background. I. Activity of T726K is dependent on
post-expression time. WT activity does not change significantly between
24 h (black) to 48 h (grey), whereas in T726K activity is slightly
lower than WT after 24 h (blue) and strongly reduced after 48 h (cyan)
when comparing c50-values.
[189]12860_2021_394_MOESM6_ESM.docx^ (1MB, docx)
Additional file 6: Figure S3. Control experiments for antibody staining
specificity and endogenous KDM6A protein levels. SW-1710 and T-24 cells
were transfected with KDM6A WT or ΔIDR variants and stained with two
different antibodies (sc-514,859, Santa Cruz biotechnology and
CST-33510, Cell Signaling) raised against epitopes within the KDM6A
central region (IDR) and 2nd antibody labeled with AbStar Red
(Abberior). Clearly, both antibodies (in red channel) detect
transfected KDM6A WT protein (very strong signals in green channel),
but not the ΔIDR variant. The untransfected control cells show
endogenous KDM6A in both cell lines stained with both KDM6A antibodies.
[190]12860_2021_394_MOESM7_ESM.docx^ (1.7MB, docx)
Additional file 7: Figure S4. Localization study of KDM6A variants in
normal and invasive urothelial (cancer) cells. A. KDM6A substitution
and deletion variants are localized in the cytoplasm and nucleus in
T-24 cells. Substitutions cause mild effects, whereas deletion variants
cause stronger cellular responses such as DNA release and mitotic
errors. B. Representative KDM6A deletion variants are localized in the
cytoplasm and nucleus in HBLAK. Two different phenotypes have been
observed: a milder phenotype (majority of cells) and a phenotype
(minority of cells) which reproducibly causes stronger cellular
responses such as DNA release and mitotic errors. DAPI counterstaining
of the nucleus and the respective overlay of both channels (DAPI in
blue and GFP in green) is displayed. Images are taken in 2048 × 2048
resolution using a widefield microscope Olympus FX81 with 60x oil
objective with post-processing using HuygensPro20.10. Effects of KDM6A
variants in SW-1710 cells is shown in Fig. [191]2.
[192]12860_2021_394_MOESM8_ESM.docx^ (1.5MB, docx)
Additional file 8: Figure S5. Western Blot control experiments and raw
blots. A. eGFP-KDM6A TPR whole cell lysate and the corresponding IP
fraction was separated on a Western Blot and stained with eGFP (CST) or
RBBP5 (CST) antibodies. RBBP5 precipitated with eGFP-KDM6A TPR. B.
Dynabeads washing control on same membrane, washed with 3x RIPA like
buffer (RLB). While KDM6A WT pulls a clearly visible RBBP5 band and a
low WDR5 band, both are undetectable in eGFP-transfected cells alone
(negative control). C. Raw blots from Fig. [193]3. Lysate and Co-IP for
selected KDM6A variants and eGFP. IP and corresponding lysate lanes
were also on the same blot. All experimental conditions for the blots
presented were kept constant. D. Raw blots from Fig. [194]1B/D. Protein
lanes shown in main figures are in red boxes. The PageRuler Plus
Prestained protein ladder (Thermo Fisher) was used for orientation.
[195]12860_2021_394_MOESM9_ESM.docx^ (54.4KB, docx)
Additional file 9: Figure S6. Pathway enrichment analysis of MS data
from KDM6A-tagGFP2 cell lines. A. Data from quadruplicates of three
independent experiments of two different KDM6A-tagGFP2 cell lines using
the reactome database. MS data sets were applied to the reactome
analysis database. The resulting output shows significantly enriched
pathways. The five most interesting pathways are depicted: cell cycle,
chromatin organization, DNA repair, RNA metabolism including rRNA
processing and protein metabolism including ribosome biogenesis and
post-translational modification of histones. B. Top ten proteins
identified by MS in KDM6A-tagGFP2 stable cell lines VM-CUB1 and RT-112.
Among the top ten proteins in KDM6A pull down and subsequent MS,
Nucleophosmin (NPM1), Nucleolin, ribosomal subunits 40S and 60S, as
well as histone variants, are detectable. Mutational KDM6A and KMT2C/D
status and experiments with RT-112 and VM-CUB1 have been recently
published [[196]18].
[197]12860_2021_394_MOESM10_ESM.docx^ (1.1MB, docx)
Additional file 10: Figure S7. Cellular back mapping identifies
co-localizing protein populations at extranuclear DNA segments. A.
images shown in Fig. [198]4, all channels. B. Analysis of KDM6A TPR and
NPM1: Cellular back mapping identifies a colocalizing population
(orange) and free KDM6A TPR (green) and NPM1 (red) at cytoplasmic DNA
segments. C. scatterplot analysis and cellular back mapping of KDM6A
variants and DAPI as indicator of KDM6A-DNA complexes. In KDM6A WT
scatterplot we identified four populations: 1 = cytoplasmic KDM6A
(without DAPI) and 4 = DAPI (without KDM6A), as well as, populations #2
and #3, which resemble different ratios of KDM6A-DNA complexes.
Population 2 is located at the lamina and in nucleolar puncta, and
population 3 is located within the nucleoplasm. Substitution variant
T726K shows minor differences in populations #2 and #4, as the lamina
in #2 is less clearly visible, but strongly enriched in population #4.
Contour scatterplot of JmjC or TPR with DAPI identified only three
populations, with population #1 being absent. Back mapping clearly
indicates compartment-wise localization of different DAPI-KDM6A JmjC or
TPR compositions.
[199]12860_2021_394_MOESM11_ESM.docx^ (1.2MB, docx)
Additional file 11: Figure S8. Distribution and expression levels of
p-γH2A.X and RAD51 after KDM6A overexpression and endogenous
localization of KDM6A during mitosis. A. p-γH2A.x protein level in
untransfected and KDM6A variants transfected T-24 cells. Note, that
even in strongly overexpressed KDM6A WT cells, p-γH2A.X was not
increased, but strongly so in KDM6A JmjC and ΔTPR cells. B. Endogenous
localization of KDM6A (CST antibody, 2nd antibody goat-anti-rabbit
AbStar red) as imaged with Leica STED SP8 in HBLAK cells. For STED, the
sample was depleted with 35% of 775 nm laser. C. RAD51 proteins were
detected at a persisting chromatin bridge of two daughter cells, with
cell 1 being KDM6A ΔTPR positive. Image was taken with Abberior STED,
excitation laser 640 nm 5% and depletion laser 775 nm with 40%.
Acknowledgements