Although the importance of JARID2 in ES cells has been established, its role in lineage‐committed cells has not been well studied, mainly because of its much lower level of expression or perceived absence (Zhang et al , 2011 ; Son et al , 2013 ). In this study, we show that, in many lineage‐committed cells including human epidermal keratinocytes, JARID2 predominantly exists as a low molecular weight (LMW) form. In the LMW form, the N‐terminal region is cleaved from full‐length JARID2 resulting in a stable C‐terminal fragment (ΔN‐JARID2). This form of JARID2 lacks N‐terminal nucleosomal and RNA binding domains (Son et al , 2013 ; Kaneko et al , 2014a ), implying a substantial effect on JARID2 functionality and its interactions with PRC2 complex. Consistent with this, a recent study showed that C‐terminal region of JARID2 cannot restore H3K27me3 marks (Cooper et al , 2016 ). We show that the level of ΔN‐JARID2 increases as differentiation of keratinocytes progresses. We find that JARID2 knockout results in impaired differentiation of keratinocytes and this effect is reversed by expression of ΔN‐JARID2, indicating that this form of JARID2 is needed for activation of polycomb target genes during differentiation.

Despite its lack of demethylase activity, JARID2 acts as an important regulator of gene expression in embryonic stem (ES) cells where it is needed for cell signalling networks necessary for maintaining the pluripotent state (Sun et al , 2008 ; Assou et al , 2009 ; Yaqubi et al , 2015 ; Sahu & Mallick, 2016 ). Consistent with this, a recent report suggests that forced expression of JARID2 alongside PRDM14, ESRRB and SALL4A can efficiently induce pluripotency in fibroblasts (Iseki et al , 2016 ). More importantly, a number of publications have shown that JARID2‐deleted ES cells either cannot differentiate or are delayed in differentiation (Peng et al , 2009 ; Shen et al , 2009 ; Landeira et al , 2015 ; Sanulli et al , 2015 ). These findings reflect a crucial role of JARID2 in early embryonic development at the onset of ES cell differentiation. Indeed, JARID2 is indispensable for normal embryonic development and its deficiency leads to deformation of several tissues in mice as well as in humans (Jung et al , 2005 ; Takeuchi et al , 2006 ; Landeira & Fisher, 2011 ). Embryos with a complete loss of JARID2 either do not survive or die soon after the birth (Jung et al , 2005 ; Takeuchi et al , 2006 ; Shen et al , 2009 ; Landeira et al , 2010 ).

The C‐terminal of JARID2 has three conserved domains (jmjN, ARID, jmjC) that are characteristic of the jumonji family of histone modifiers (Fig 1 A), which catalyse demethylation of histones. The C‐terminal ARID domain of JARID2 is required for DNA binding. In addition, JARID2 C‐terminus also has a zinc finger domain, which is needed for its interaction with SUZ12, another component of PRC2 (Peng et al , 2009 ). The jmjC domain is required for demethylase activity in other jumonji family members. However, two amino acid changes in JARID2's demethylase domain are thought to render it inactive (Klose et al , 2006 ; Landeira & Fisher, 2011 ).

JARID2 is required for recruitment of PRC2 to chromatin in embryonic stem cells (Peng et al , 2009 ; Shen et al , 2009 ; Landeira et al , 2010 ; Li et al , 2010 ; Pasini et al , 2010 ; Landeira & Fisher, 2011 ; Holoch & Margueron, 2017 ). Multiple studies in mouse and human show that N‐terminal region of JARID2 (Fig 1 A) is required for PRC2 recruitment and modulation of PRC2 activity (Cooper, Son et al , 2013 ; Kaneko et al , 2014a ; da Rocha et al , 2014 ; Sanulli et al , 2015 ; Grijzenhout et al , 2016 ). The N‐terminal region consists of a nucleosomal binding domain and a RNA binding domain that together modulate PRC2 binding to genomic DNA (Son et al , 2013 ; Kaneko et al , 2014a ; da Rocha et al , 2014 ). In addition, recently it has been shown that this region of JARID2 is needed for recruitment of PRC2‐ to PRC1‐modified nucleosomes (Cooper et al , 2016 ). It is clear from multiple studies that removal of JARID2 results in reduced occupancy of PRC2 on chromatin (Peng et al , 2009 ; Shen et al , 2009 ; Landeira et al , 2010 ; Li et al , 2010 ; Pasini et al , 2010 ; Landeira & Fisher, 2011 ). But surprisingly, JARID2 removal does not result in significant and consistent changes in H3K27me3 levels (Landeira & Fisher, 2011 ). Although in some studies JARID2 depletion in ES cells is observed to decrease H3K27me3 levels (Landeira et al , 2010 ; Pasini et al , 2010 ), other studies have reported either no change (Peng et al , 2009 ) or increased levels of H3K27me3 upon JARID2 removal (Peng et al , 2009 ; Shen et al , 2009 ). Further adding to this lack of clarity on the role of JARID2 in modulation of PRC2 activity, in in vitro studies JARID2 appears to inhibit (Peng et al , 2009 ; Shen et al , 2009 ) as well as activate (Li et al , 2010 ) the methyltransferase activity of EZH2. It has been suggested that JARID2's N‐terminal domain interacts with RNAs as well as nucleosomes (Son et al , 2013 ; Kaneko et al , 2014b ) and its post‐translational modifications determine its effect on PRC2 activity (Sanulli et al , 2015 ). A recent study has also shown that, in mouse ES cells, JARID2 can modulate PRC2 activity through its interaction with another histone methylase, setDB1 (Fei et al , 2015 ). JARID2‐setDB1 interaction has also been identified in lineage‐committed cells including lymphocytes (Macian et al , 2002 ; Pereira et al , 2014 ) and cardiomyocytes (Mysliwiec et al , 2011 ) where JARID2 is shown to modulate other histone modifications such as H3K9me3.

Polycomb group (PcG) proteins are very important transcriptional repressors and play a crucial role in regulating gene expression during development (Margueron & Reinberg, 2011 ; Holoch & Margueron, 2017 ). They function by catalysing histone modifications that result in repressive chromatin and down‐regulation of neighbouring genes. Polycomb group proteins form two major complexes, polycomb repressive complex‐1 (PRC1) and polycomb repressive complex‐2 (PRC2). PRC2 functions by catalysing trimethylation of histone H3 at lysine 27 (H3K27me3; Simon & Kingston, 2013 ). Polycomb repressive complex‐2 consists of four core proteins, SUZ12, EED, RbAp46/48 and the catalytic subunit EZH2. At the molecular level, how PRC2 is recruited to its sites of action is not yet completely clear. Recent proteomic studies have revealed that PRC2 transiently associates with many proteins such as MTF2, EPOP, AEBP2 and JARID2 that typically interact with PRC2 in a mutually exclusive fashion resulting in different subclasses of PRC2 (Kim et al , 2009 ; Peng et al , 2009 ; Shen et al , 2009 ; Landeira et al , 2010 ; Li et al , 2010 ; Pasini et al , 2010 ; Walker et al , 2010 ; Casanova et al , 2011 ; Landeira & Fisher, 2011 ; Beringer et al , 2016 ; Grijzenhout et al , 2016 ; Liefke et al , 2016 ; Holoch & Margueron, 2017 ). Although the molecular roles of many of these interacting proteins are not well understood, many of them can modulate enzymatic activity or recruitment of PRC2 to chromatin (Holoch & Margueron, 2017 ).

Results

A low molecular weight form of JARID2 exists in lineage‐committed cells JARID2 has been extensively studied in embryonic stem (ES) cells where it is reported as a 140 kDa protein (Fig 1B). It is thought that JARID2 is not expressed or is expressed at very low levels in lineage‐committed cells (Zhang et al, 2011; Son et al, 2013). Therefore, we investigated JARID2 mRNA as well as protein expression in multiple types of lineage‐committed human cells (Fig 1B and C). In most cell types, JARID2 mRNA is present at detectable but lower levels than in the ES cells (Fig 1C). Surprisingly, when we investigated protein levels in lineage‐committed cells (Fig 1B), we detected another band at around ~80 kDa that has not been reported previously. We observed that in the majority of cell types we studied, this band is much more dominant than the 140 kDa band corresponding to the canonical full‐length JARID2 isoform‐1 (Fig 1B). To verify that this low molecular weight form is encoded by JARID2 and is not a non‐specific band cross‐reacting with our antibody, we transfected keratinocytes (HaCaT cells), HEK293T and K562 cells (Figs 1D and EV1A and B) with different JARID2 siRNAs designed to target the 5′ (exon 3) and 3′‐ends (exon 15) of JARID2 mRNA (Appendix Table S1). Transfection of both siRNAs resulted in disappearance of the ~80kDa band along with the canonical 140 kDa band (Figs 1D and EV1A and B). The siRNA‐mediated knockdown and Western blot with an alternative JARID2 antibody (Fig EV1B) confirmed that this is a low molecular weight (LMW) form of JARID2. To rule out the possibility that this band is a degradation product of JARID2, we also extracted protein in the presence of increasing amount of protease inhibitor and observed no difference in the levels of the LMW form (Fig EV1C). Click here to expand this figure. Figure EV1.The 80 kDa band is a JARID2 product Densitometric measurements of the 80kDa band detected in HaCaT and HEK293T cell lysates (corresponding to immunoblots in Fig 1D). Normalised levels of the 80kDa band with respect to GAPDH after transfecting HaCaT and HEK293T cells with non‐silencing siRNA control (unfilled bars) and two independent siRNAs (black bars) are shown. Means and SE were calculated based on three independent experiments for HaCaTs. Significance (***P < 0.001) was calculated using one‐way ANOVA in comparison with non‐silencing control. Means were calculated for two independent experiments for HEK293T cells. Immunoblots showing the 80kDa band are recognised by two separate JARID2 antibodies. K562 cells transfected with control and JARID2 siRNA1 were blotted with a monoclonal anti‐JARID2 antibody (D6M9X, left hand blot), stripped and re‐probed with a polyclonal anti‐JARID2 antibody (GTX129020, right hand blot). Both antibodies detect the 140 and 80 kDa bands and their reduced expression when transfected with JARID2 siRNA confirms that these are JARID2 bands. The polyclonal antibody is less effective at detecting JARID2 and a non‐specific band around 100 kDa was also detected. Immunoblot of protein samples from K562 cells extracted in the presence of increasing amounts of protease inhibitor. No change was observed in either the 80 kDa or 140 kDa band confirming that 80 kDa band is not a degradation product. Source data are available online for this figure.

LMW JARID2 is not a transcriptional isoform Next, we sought to understand whether the LMW form is translated from a distinct transcript of JARID2. According to latest ENSEMBL annotations of human genes, three different mRNA isoforms of JARID2 have been predicted (Fig 2A; Rosenbloom et al, 2015). According to size predictions, the three isoforms would produce proteins of sizes 140, 120 and 106 kDa, much larger than ~80 kDa indicating that this LMW form might not correspond to one of the annotated isoforms of JARID2. In addition, the mRNA for isoform‐3 does not express exon 15, which is targeted by one of the siRNAs used in our knockdown experiment (Figs 1D and 2A), indicating that the ~80 kDa form is not a product of isoform‐3. To test whether it might be a product of a mRNA variant transcribed from an internal promoter that is not yet part of current annotations, we analysed a large collection of CAGE (cap analysis gene expression) tag data that is available through the ENCODE database (Rosenbloom et al, 2013). In CAGE analysis, short fragments from 5′ ends of capped RNAs are sequenced and mapped back to the genome to find transcription start sites. CAGE tag data clearly indicate that in most of the cell types we tested (Fig 1B), JARID2 is predominantly transcribed from single transcription start site matching that of JARID2 isoform‐1 (Fig 2B). We also designed primers to specifically amplify each isoform of JARID2 (Appendix Table S2) and carried out qPCR analysis to detect levels of the three different isoforms. Our qPCR measurements further confirmed that isoform‐1 is the predominant isoform in these cells (Fig 2C). To rule out the possibility that the ~80 kDA band is a product of an mRNA with same transcription start site as isoform‐1, but with a different splicing pattern, we amplified JARID2 mRNA using RT–PCR with primers in exon 3 and exon 15 (Appendix Table S3) which, according to our knockdown experiments, are part of mRNA that produces this low molecular weight isoform (Fig 1D). Using these primers, only one product was amplified and its size as well as sequence confirmed that it contained all the exons between exon 3 and exon 15 (Fig 2D, see Appendix). In addition, RT–PCR using primers in the first and the last exon also amplified only one product of the expected size (Fig 2D). This confirmed that the LMW form of JARID2 is a product of mRNA with a complete set of exons as in the case of full‐length mRNA of isoform‐1. This suggested that LMW JARID2 is either a cleaved protein product of full‐length JARID2 or is translated from an internal site that is distinct from the isoform‐1 translation site. Figure 2.JARID2 LMW is a cleaved product of full‐length JARID2 isoform‐1 A schematic showing three different isoforms of human JARID2, as predicted by ensembl annotations. The translation start site of each isoform is labelled using green arrow. Predicted sizes (140, 120 and 106 kDa) of proteins corresponding to three isoforms have been indicated. CAGE‐seq data showing the transcription start sites of JARID2 in different cell lines. CAGE peak is mainly observed at JARID2 isoform‐1 in most cell types. JARID2 gene structure is shown at the top. qPCR measurements of RNA levels (n = 3) for the three isoforms of JARID2 in HaCaT cells. The RNA levels are plotted relative to 18S rRNA. Level of JARID2 isoform‐1 is significantly higher than that of isoform‐2 (***P < 0.001) and isoform‐3 (****P < 0.0001). Data for three independent experiments are represented as mean ± SE. Multiple comparisons were performed and P‐values were calculated using one‐way ANOVA. Agarose gel showing RT–PCR products amplified using primers in exon 3 and exon 15 (2.9 kb) as well as RT–PCR product corresponding to JARID2 isoform‐1 (3.7 kb) amplified using the first (exon 1) and the last exon (exon 18). Only one product at the right size is observed indicating that it is unlikely that JARID2 might be a product of an alternatively spliced isoform of JARID2. Ribosomal profiling data mapped on JARID2 isoform‐1 and isoform‐2. Main translation site corresponding to isoform‐1 is highlighted using a dashed box. CRISPR‐Cas9 knockout of JARID2 using a sgRNA guide designed to target main translation start site as seen in (E). Immunoblot revealed that ˜80 kDa form was removed from JARID2 KO lines. Densitometric measurements corresponding to the experiment are shown in Fig EV2D. Immunoblot after a full‐length JARID2 isoform‐1 was expressed from an exogenous vector in HaCaT cells showed an increase (2‐ to 3‐fold) in ˜80 kDa level. Densitometric measurements are shown in Fig EV2E. An immunoblot with exogenous expression of 140kDa band in a control cell line is shown in Fig EV2F. Immunoprecipitation using anti‐JARID2 antibody and followed by mass spectrometry identification of the ˜80 kDa band detected JARID2 peptides spanning only C‐terminus of JARID2. The identified peptides are shown as red arrows on schematic of JARID2 sequence. Source data are available online for this figure. Source Data for Figure 2 [embj201798449-sup-0004-SDataFig2.pptx]

LMW JARID2 is a cleaved product of isoform‐1 To check whether JARID2 mRNA is translated from two distinct translation start sites, one corresponding to the reported 140 kDa product and other corresponding to the ~80 kDa low molecular weight form, we analysed published ribosome profiling data (Michel et al, 2015). We could only identify a single ribosome initiation site, which corresponded to the 140 kDa product (Fig 2E). Taking into consideration the above observations and the fact that we used a JARID2 antibody that recognises the C‐terminal region of JARID2, we can predict that a ~80 kDa protein can be produced from translational start site which is far downstream of translational start site of isoform‐1. However, if LMW JARID2 is a cleaved product of full‐length JARID2, rather than a distinct isoform translated from an internal start site, mutating the translation start site of JARID2 isoform‐1 should also knockdown LMW JARID2. To test this, we knocked out isoform‐1 by CRISPR/Cas9‐mediated targeting of its translation start site (Fig EV2A and B). In knockout (KO) cells, where we targeted the translation start site of isoform‐1, we detected the same level of mRNA as wild type (Fig EV2C). However, the LMW JARID2 protein band disappeared (Figs 2F and EV2D). This supports the hypothesis that the 80 kDa LMW form and JARID2 isoform‐1 have identical translation start sites and that the LMW form is a cleavage product of JARID2 isoform‐1. Click here to expand this figure. Figure EV2.CRISPR/Cas9‐mediated deletion of JARID2 Diagram showing location of sgRNA sequence in the JARID2 loci. Red sequence depicts the PAM and red arrow shows the site of double strand cleavage by Cas9 nuclease. The DNA sequences of JARID2 targeted alleles show five bases deletion in one copy and a one base insertion in the other copy, resulting in a frameshift in the open reading frame. JARID2 mRNA levels measured using qPCR in wild‐type (WT) and JARID2 knockout (KO1 and KO2) cells. Data from at least three independent experiments (n = 3) are represented as mean ± SE. Densitometric measurements (corresponding to immunoblots in Fig 2F) of the 80 kDa band in wild‐type (WT) and two JARID2 knockout lines (KO1, KO2) relative to GAPDH levels. Data from three independent experiments (n = 3) are represented as mean ± SE, and significance was calculated in multiple comparison using one‐way ANOVA (*P < 0.05). Densitometric measurements (corresponding to immunoblots in Fig 2G) showing an increase in 80kDa band levels relative to GAPDH in cells transfected with full‐length JARID2 (FL‐JARID2; red) compared to empty vector control (blue). Data from three independent experiments (n = 3) are represented as mean ± SE and significance is calculated using t‐test (**P < 0.01). Immunoblot of HEK293T cells transfected with FL‐JARID2 compared to empty vector control. The blot clearly shows an increase in the levels of the 140 kDa band corresponding to canonical JARID2 indicating expression of the correct protein. The blot also shows an increase in the 80 kDa band. Immunoblot with anti‐Flag antibody showing that the Flag‐tagged N‐terminal fragment of FL‐JARID2 is not detected in HaCaT cell lysates. Source data are available online for this figure. To further verify that the LMW form is indeed a cleavage product of JARID2 isoform‐1, we transfected HaCaT cells with N‐terminally flag‐tagged ORF of full‐length JARID2 (FL‐JARID2) isoform‐1 expressing plasmid vector. If LMW JARID2 is truly derived from full‐length JARID2, we predicted that exogenous expression of full‐length JARID2 would also result in an increase in the levels of the LMW form. Consistent with this hypothesis, we observed that LMW levels increased 2‐ to 3‐fold upon exogenous expression of full‐length JARID2 (Figs 2G and EV2E and F). However, in a blot with anti‐Flag antibody, which should detect the N‐terminal tag on JARID2, we could not detect the full‐length JARID2, 80 kDa band or any other low molecular weight product (Fig EV2G), indicating that the N‐terminal portion of JARID2 is missing in these cells. To further confirm this, we carried out mass spectrometry identification of the LMW band. In this experiment, we detected peptides spanning only the C‐terminal of JARID2 (Figs 2H and EV3). All these observations strongly support the hypothesis that the LMW form is a cleaved product of full‐length JARID2 isoform‐1 and is missing the N‐terminal portion. Accordingly, we designated the LMW form ΔN‐JARID2. To confirm the size of ΔN‐JARID2 and to estimate the cleavage position, we prepared plasmid constructs expressing different length C‐terminal fragments of JARID2 (Appendix Fig S1A) and checked if their predicted protein products (103, 88 and 79 kDa) run at a similar size to ΔN‐JARID2 (Appendix Fig S1B). We found that the fragment spanning 554‐1,246 amino acids produced a protein product (79 kDa) that co‐migrated with ΔN‐JARID2 confirming that the size of ΔN‐JARID2 is ~80 kDa. This is also consistent with the mass spectrometry data where the N‐terminal‐most peptide is detected at amino acid position 589 of JARID2 sequence (Fig EV3). Click here to expand this figure. Figure EV3.Mass spectrometry identification of 80kDa band Silver staining of a protein sample electrophoresed on a polyacrylamide gel after immunoprecipitation with JARID2 antibody (D6M9X). The ˜80 kDa band was cut and subjected to mass spectrometry analysis. JARID2 peptides detected in mass spectrometry analysis of ˜80 kDa band and their position on JARID2 sequence.

ΔN‐JARID2 is required for cell differentiation Previous studies have shown that PRC2 and JARID2 play an important role in epidermal development and differentiation (Ezhkova et al, 2009, 2011; Mejetta et al, 2011; Wurm et al, 2015). Therefore, we explored the role of ΔN‐JARID2 in epidermal differentiation. First, we examined expression of ΔN‐JARID2 in keratinocytes (HaCaTs), which represent a good model for human epidermal differentiation (Wilson, 2014). We differentiated HaCaTs by growing them to confluency in low calcium medium for 4 weeks before switching to high calcium medium for up to 6 days (Fig 3A). We confirmed differentiation by measuring levels of epidermal differentiation markers such as involucrin (IVL) and trans‐glutaminase1 (TGase1) on days 0, 1, 3 and 6 after switching to high calcium medium (Fig 3B). Interestingly, as differentiation progressed, ΔN‐JARID2 levels also increased up to 2‐fold (Fig 3C). Therefore, we speculated that ΔN‐JARID2 might be important for HaCaT differentiation. Figure 3.Effect of JARID2 knockout on differentiation markers The calcium‐induced differentiation protocol used in this study. Up until day 0, cells were maintained in low calcium media and then induced to differentiate by growing them in high calcium medium for 6 days. Cells were harvested for immunoblot at day 0, day 1, day 3 and day 6 of differentiation. Immunoblot showing increase in expression levels of differentiation markers involucrin (IVL) and Transglutaminase‐1 (TGase‐1) as differentiation progressed indicating that keratinocyte differentiation protocol was successful. Protein levels on day 0 (D0), day 1 (D1), day 3 (D3) and day 6 (D6) of differentiation are shown. Immunoblot for JARID2 during D0, D1, D3 and D6 of differentiation (as in B) are shown. Effect of JARID2 removal on levels of differentiation markers involucrin (IVL), keratin‐1 (KRT1) and keratin‐10 (KRT10) mRNAs as measured in qPCR experiment relative to 18S rRNA and the rescue using exogenous expression of ΔN‐JARID2. The effect of exogenous expression of full‐length JARID2 (FL‐JARID2) in JARID2 knockout is also shown. Data from wild‐type HaCaTs, two independent JARID2 knockout (KO1 and KO2) HaCaT lines and KO cells exogenously expressing empty vector control, ΔN‐JARID2 and FL‐JARID2 are shown. Data from three independent experiments (n = 3) are represented as mean ± SE, and multiple comparisons were performed using one‐way ANOVA (****P < 0.0001, ***P < 0.001, **P < 0.01 and *P < 0.05). Levels of involucrin (IVL), keratin‐1 (KRT1) and keratin‐10 (KRT10) mRNAs as above but measured on day 3 of differentiation. Source data are available online for this figure. Source Data for Figure 3 [embj201798449-sup-0005-SDataFig3.pptx] To test this hypothesis, we compared differentiation in JARID2‐null HaCaTs and wild‐type HaCaTs by comparing RNA levels of differentiation markers keratin‐1, keratin‐10 and involucrin on day 0 and day 3 of differentiation (Fig 3D and E). We predicted that removal of JARID2 should result in de‐repression of differentiation genes. However, in JARID2‐null cells, we observed significant down‐regulation of involucrin and keratin‐1 genes, which are earlier reported to be polycomb targets in Keratinocytes (Sen et al, 2008) (Fig 3D and E). Given that ΔN‐JARID2 is the main form of JARID2 in keratinocytes and its level increases during differentiation, we speculated that the impaired differentiation seen in JARID2‐null cells is due to removal of the ΔN‐JARID2 form rather than the full‐length form of JARID2. In such a case, exogenous expression of ΔN‐JARID2 should be sufficient to rescue down‐regulation of differentiation markers. We therefore transfected JARID2‐null cells with plasmids expressing the C‐terminal ~80 kDa fragment (Appendix Fig S1). Since we are yet to identify exact cleavage site of JARID2, this C‐terminal fragment should mimic the ΔN‐JARID2 form. Significantly, expression of the C‐terminal ~80 kDa fragment was sufficient to rescue the expression of differentiation markers, indicating that the effect of JARID2 knockout on differentiation is most likely due to ΔN‐JARID2 (Fig 3D and E). As wild‐type cells express low levels of full‐length JARID2, it could be argued that the impaired differentiation in JARID2 KO might be a combined effect of both full‐length and ΔN‐JARID2. To rule out this possibility, we also studied the effect of exogenously expressed full‐length JARID2 (FL‐JARID2). However, on day 3, expression of full‐length JARID2 leads to suppression of differentiation markers (Fig 3E). This supports a role for ΔN‐JARID2 in promoting differentiation, whereas full‐length JARID2, like other polycomb proteins, functions to suppress differentiation.