An Essay on the Polycomb group proteins

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Introduction

The polycomb group proteins are extremely important in regulation of gene transcription. They have been shown to regulate the repression of HOX genes in insects and mammalian cells and have also been implicated in the direction of differentiation in pluripotent cells (Schuettengruber et al., 2007). It is argued that in the core complex of the Polycomb Repressive Complex 2 there is a group of 4 or 5 of these polycomb group proteins make up a complex called the Polycomb Repressive Complex 2. This is a complex that is responsible for trimethylating Histone 3 at lysine 27, which is a mechanism that allows the histones to associate with each other in a much tighter complex and leaves the chromatin, and ultimately the gene of interest, inaccessible to transcription factors. This causes a repression of that gene expression because transcription factors are unable to bind and RNA polymerase is never recruited to the gene promoter (Guenther and Young, 2010).

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The most important protein in this complex is EZH2, which is the histone methyltransferase that is responsible for the catalytic activity of the Polycomb repressive complex 2. Mutations in EZH2 have shown that it is essential for normal cellular functions; when mutated in a cell, the cell generally shows many cancerous phenotypes. Although it is hard to pinpoint the exact mechanism for these cancerous properties, it is generally believed that the regulation of gene suppression through Polycomb Repressive Complex 2 and EZH2 is extremely important to most cell types (Sneeringer et al., 2010). In embryonic stem cells, which are pluripotent cells, the deregulation of Polycomb Repressive Complex 2 or EZH2 causes abnormal differentiation or an absence of differentiation altogether (Peng et al., 2009).

In the context of immunology, there is possible epigenetic regulation of important proteins of the immune system, most notably Major Histocompatibility Complex Class II and CIITA. Major Histocompatibility Complex Class II is important to many cells of the body, because it serves as the peptide presentation site on antigen presenting cells of the immune system, although most cells in the human body can be stimulated to produce and display this protein on the cell surface through cytokines such as Interferon gamma. Cells that display Major Histocompatibility Complex class II on the cell surface use this protein to display short peptides of foreign protein that are gathered from the extracellular environment. When an immune cell, such as a B cell or T cell, recognizes this peptide on the Major Histocompatibility Complex II molecule as foreign, it will activate the immune cell and produce a response specific to that foreign peptide through a very complex cascade of events. Needless to say, this protein is extremely important in the context of immunity. CIITA is known as the Class II Transactivator, and is an important transcription factor for the Major Histocompatibility Complex Class II protein. CIITA, which does not bind DNA directly, is part of a larger complex of transcription factors known as the enhanceosome complex, and uses the other proteins to bind to the DNA. Once it binds to the Major Histocompatibility Complex Class II proximal promoter, transcription of this protein can begin. There are four promoters for CIITA found in human cells, but promoter IV is the most important when studying cells that are not professional antigen presenting cells, as those cells (such as dendritic cells) use other promoters for CIITA expression. (Ting and Trowsdale, 2002).

Major Histocompatibility Complex Class II is important for the immune system to recognize pathogens, but its regulation of expression on cell surface is also important. It is understood that the ability of cancerous cells to move about the human body unnoticed is that they are able to escape the immune system. This is because they are able to hide from immune surveillance, which is heavily reliant upon Major Histocompatibility Complex II and antigen presentation. Cancerous cells generally have mutations in DNA, which usually end up in mutated proteins. The immune system has evolved very specific mechanisms to recognize these mutated proteins as foreign proteins. Normally, as soon as a cell begins to act abnormally and produce these mutated proteins or not produce these proteins at all, the immune system can recognize these peptides displayed on the surface through Major Histocompatibility Complex Class II. But, an important observation in cancerous cells is that many of these tumors downregulate Major Histocompatibility Complex Class II, and are therefore able to "hide" from the immune system. (Wright and Ting, 2006).

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Unpublished data in the Greer lab has shown that Histone 3 Lysine 27 trimethylation is extremely dynamic in cells when CIITA transcription is turned on and off. Upon stimulation to display Major Histocompatibility Complex II by interferon gamma, cells dramatically downregulate the trimethylation of H3K27me3. This is in coordination with an opening up of the promoter for transcription availability. Also, the opposite effect is shown in cells that do not constitutively express Major Histocompatibility Complex II and are not stimulated to produce this protein; high levels of this repressive mark are found at the CIITA promoter. This in turn would suggest that this histone repressive modification is what is in control of the expression of this protein. This would also suggest that the Polycomb Repressive Complex 2 and EZH2 catalytic activity at CIITA pIV is in control of the expression of this protein. Knowing this, it would be very beneficial to understand the mechanism by which Polycomb Repressive Complex 2 is recruited to the promoter.

Although much is known about polycomb group proteins and the polycomb repressive complex 2, it is still unclear what sequences of DNA the polycomb complex is recruited to; PREs, or polycomb responsive elements are the homologous sequences on that the Polycomb Repressive Complex 2 complex binds with the help of a recruiter protein. These are studied and relatively well-known in Drosophila, but is a topic of continued debate in mammalian cells (Simon and Kingston, 2009.) There are no DNA binding motifs in any of the members of the polycomb repressive complex 2, and therefore it is thought that this complex does not directly bind to DNA. Taking this in mind, it is currently a topic of much speculation as to how this complex is recruited (Villa et al., 2007).

A possible suspect for the recruit of Polycomb Repressive Complex 2 to the gene promoter is JARID2. JARID2 is a DNA binding protein that is enriched in pluripotent cells. It has an ARID domain that is thought to be the DNA binding domain of the protein. It has also been found that not only do Polycomb Repressive Complex 2/JARID2 gene targets contain many CCG repeats in the DNA, it has been shown that similar proteins in the same family as JARID2 have ARID domains that preferentially bind to CCG repeats in DNA. This paper addresses the importance of JARID2 and it is hypothesized that JARID2 is an important partner in the binding of the Polycomb repressive complex to target genes.

Experimental Results

In Figure 1, the paper hypothesizes that JARID2 is a component of the Polycomb Repressive Complex 2 complex. Using HeLa cells that express a FLAG-HA-SUZ12 protein, an immunoprecipitation was performed to pull down anything that is associating with SUZ12, because this is a known component of the Polycomb Repressive Complex 2 complex. If anything is associated with SUZ12, it can be assumed that the proteins are associating with the Polycomb repressive complex. Once the proteins are immunoprecipitated, mass spectrometry was run on the proteins to determine what is associating with SUZ12. The proteins are identified using a mass spectrometer and then the properties of the proteins identified, including molecular weight, are put in a database to compare against other known proteins in biological systems. JARID2 was found to be one of the proteins associating with SUZ12 (Figure 1a). This is important to note because SUZ12 is one of the core Polycomb Repressive Complex 2 proteins that make up the complex. A 293T cell line was grown up to stably express Flag-HA-JARID2 and another immunoprecipitation was performed to identify the proteins associating with JARID2. As a result, all of the Polycomb Repressive Complex 2 proteins were pulled down with JARID2 (Figure 1b). This can be interpreted that JARID2 is associating with all of the known members of the Polycomb Repressive Complex 2 complex. When Flag-HA-JARID2 is overexpressed in embryonic stem cells, it associates with the endogenous components of the Polycomb Repressive Complex 2 but does not associate with Ring1b, which is a part of the PRC1 complex (Figure 1c). This is important to note because this differentiates JARID2 as associating with only the Polycomb Repressive Complex 2 complex, not both PRC1 and 2. Finally, in mouse embryonic stem cells, endogenous JARID2 associates with endogenous SUZ12 and EZH2, which both are part of the Polycomb Repressive Complex 2 complex. The overall conclusion from the figure is that JARID2 associates with Polycomb Repressive Complex 2 complex and most importantly, in endogenous embryonic stem cells, not just in overexpressed protein situations. This is indicative of an in vivo system, and that the JARID2 associates with Polycomb Repressive Complex 2 in the cell in a normal cell system.

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In Figure 2, the paper hypothesizes that while knowing JARID2 possesses transcriptionally repressive properties, it is unable to repress genes without the Polycomb Repressive Complex 2 complex. Several mutants of the JARID2 protein were created and tested for binding to Polycomb Repressive Complex 2. It was found that mutating a small region, amino acids 147-165, negated Polycomb Repressive Complex 2 binding. This is the same region that is considered the repressive region of the JARID2 protein, indicating that it is possible that Polycomb Repressive Complex 2 binding is important for the repressive functions of JARID2 (Figure 2a). A luciferase assay was performed using a Gal4/JARID2 construct (Figure 2b) with a WT JARID2 protein and the JARID2 protein with the Polycomb Repressive Complex 2 binding domain mutation that were stably expressed in 293T cells. It was found that the WT JARID2 retained its repressive function, while the Polycomb Repressive Complex 2 binding mutation JARID2 protein lost its repressive function and luciferase activity was on the same level as the positive control (Figure 2c). This means that if the luciferase activity is observed, the promoter is no longer repressed. When a chromatin immunoprecipitation was performed on these same cells with the WT and mutant JARID2 proteins, it was found that EZH2 and SUZ12 were recruited to the luciferase promoter in the WT JARID2 cells. When the mutant was introduced, the recruitment of these proteins was significantly decreased, indicating that the Polycomb Repressive Complex 2 complex was not bound to the promoter. The RING1B protein showed similar results, being recruited to the luciferase promoter in the WT cells, but a loss of recruitment in the mutant JARID2 cells. This is in line with current opinions that model Polycomb Repressive Complex 2 to be dependent on PRC1 to bind. To look at downstream effects of Polycomb Repressive Complex 2 binding and further prove the loss of recruitment, Histone 3 Lysine 27 trimethylation repressive marks were observed. In the wild type, the marks were high, indicating a repressive effect of wild type JARID2 and Polycomb Repressive Complex 2 binding, while the mutant showed a significant loss in the repressive mark. Histone 3 lysine 4 trimethylation was also observed, and the opposite effect was observed. Taking all of this, it is clear that JARID2 cannot possess its transcriptionally repressive properties without the help of Polycomb Repressive Complex 2 and its binding to the target gene. It also shows that JARID2 must be associated with Polycomb Repressive Complex 2 for gene repression in this manner.

In figure 3, the paper hypothesizes that JARID2 is a regulator of the binding of Polycomb Repressive Complex 2 to target genes. A ChIP-sequence in mouse embryonic stem cells was performed to determine the binding sites of JARID2 and SUZ12, and EZH2. It was found that both JARID2 and SUZ12 bind at gene promoter regions of the DNA about 70 percent of the time (Figure 3a). When looking at specific binding sites, SUZ12 is bound to 90 percent of JARID2 binding sites (Figure 3b). Looking at EZH2 as well, most of JARID2 binding sites are also occupied by EZH2 as well as SUZ12 (Figure 3c). Interestingly, when JARID2 is knocked down with shRNA, 98 percent of the genes that SUZ12 is bound to in normal cells are decreased at least by 3 fold (Figure 3d). This is indicative of the dependence of SUZ12 on JARID2 DNA binding. Further evidence for Polycomb Repressive Complex 2 association, JARID2 associates with H3K27me3 marks in ES, which is a hallmark of Polycomb Repressive Complex 2 suppression (Figure 3e). Next, the ARID domain of the JARID2 protein was looked at for possible involvement in recruiting Polycomb Repressive Complex 2 to target promoters. To investigate this, endogenous JARID2 was knocked down by shRNA and ES cells were transiently transfected with Flag-JARID2 wild time and Flag-JARID2 ARID mutant (Figure 3f). The transient expression of the wild type JARID2 protein, after the knockdown of endogenous JARID2, was able to partially restore the binding of EZH2 and SUZ12 (Polycomb Repressive Complex 2 complex) to the promoters of two genes in ES cells, but the JARID2 ARID mutant was not able to recruit these proteins to the promoter. This suggests that the ARID domain of the JARID2 protein is responsible for regulating Polycomb Repressive Complex 2 binding to gene promoters. Lastly, a ChIP was performed in ES with shRNA for SUZ12 and JARID2 to determine if JARID2 is stable on the promoter without the Polycomb Repressive Complex 2 complex or if it must be stabilized by Polycomb Repressive Complex 2. The results showed that when JARID2 is knocked down, there is less bound SUZ12 to the promoter, as expected, and when SUZ12 is knocked down, there is notably less JARID2 bound to the promoter. This indicates that the Polycomb Repressive Complex 2-JARID2 complex is recruited to the promoter at the same time, as one complex. JARID2 does not bind to the DNA without the Polycomb Repressive Complex 2. All of this together shows that JARID2 forms a stable association with Polycomb Repressive Complex 2 and is recruited to the DNA as one unit.

In figure 4, the paper explores the hypothesis that JARID2 is essential in the ES cell differentiation process, and without this there is no differentiation. First, ES cells were stably transfected with control shRNA and JARID2 shRNA to knock down endogenous JARID2 (Figure 4b). From this, the cells were stimulated by embryoid bodies to differentiate. There were no defects in the JARID2 knockdown cell proliferation, but they did not properly differentiate for a prolonged period of time. After 10 days, there was notably less differentiation than in the control shRNA ES cells (Figure 4a). Quantitative PCR was performed on these cells at several differentiation marker genes and two non-differentiation (pluripotent) marker genes. In the JARID2 shRNA cells, the amount of pluripotent gene transcription was slightly more than the control shRNA and the amount of differentiation gene transcription was significantly less than the control shRNA after 10 days of proliferation. A genome expression analysis was used to analyze the difference in the amount of upregulated and downregulated differentiation genes in the shRNA control and the JARID2 shRNA. Overall there was much less upregulated and downregulated genes in the JARID2 shRNA cells. All of this shows that JARID2 is essential for proper ES cell differentiation.

Supplementary Data

In supplementary Figure 1, it adds support for the JARID2 protein associating with the Polycomb Repressive Complex 2. Hela cells were made to express the Flag-SUZ12 protein and a western blot performed to check expression (Figure S1a). 293T cells were transfected with Flag-JARID2 protein and an immunoprecipitation of Flag-JARID2 was performed to check the expression of JARID2 and the association of SUZ12 (Figure S1b). Endogenous protein association was checked after transfection of HA-JARID2 and IP of HA to check association of JARID2 with endogenous SUZ12 and EZH2 in 293T cells (Figure S1c). The same cells expressing the HA-JARID2 immunoprecipitated with SUZ12 and EZH2 antibodies and western blotted with antibodies for SUZ12 and EZH2 to check for in vivo association of protein (Figure S1d). ES were transiently transfected HA-JARID2 and western blots were performed to show the slower migration of the HA-JARID2 rather than the endogenous. They were then western blotted with SUZ12 and EZH2 antibodies (Figure S1e). Finally, the cells were introduced to varying salt concentrations and IPed with EZH2 antibody and western blotted for EZH2, SUZ12, and JARID2. This indicates that the complex is stable in high salt concentrations (Figure S1f).

In supplementary Figure 2, there is further support of JARID2 interaction with Polycomb Repressive Complex 2 complexes by size exclusion chromatography and western blotting (Figure S2a). Also, using insect cell extracts with transfection of FLAG-JARID2 and IP for FLAG, western blots were performed with antibodies for EZH2, SUZ12, and EED. In supplementary Figure 3, JARID2 was localized to PCR2 bodies using immunofluorescence of HA-JARID2 and antibodies for SUZ12 and EZH2. RING1B is part of the PRC1 (Figure S3a). Different domains of JARID2 were mutated and then western blotted for SUZ12 and EZH2 to determine the necessary domains for binding of JARID2 to Polycomb Repressive Complex 2 components (Figure S3b). Luciferase assay was performed to determine the repressive activity of the mutant JARID2 (Figure S3c).

In supplementary Figure 4, many examples of results from the ChIP-sequencing assay performed were shown. This shows that many of the JARID2 binding sites were also binding sites for SUZ12, further indicating the interaction between JARID2 and Polycomb Repressive Complex 2 components. In supplementary Figure 5, there is diagrams representing the pathways that JARID2 affects downstream, and the comparison of the Polycomb Repressive Complex 2 component's downstream repressive effects. This again, is further evidence of the association between JARID2 and the Polycomb Repressive Complex 2 (Figure S5a-d). A western blot showing the successful knockdown of JARID2 protein in the experiment in Figure 3 with shRNA (Figure S5e).

Supplementary Figure 6 provides further evidence that PRC2 requires JARID2 to be bound to DNA in order for PRC2 to bind to target genes. A knockdown of JARID2 shows less binding of PRC2 component proteins bound to promoters. Supplementary Figure 7 shows the downstream effects of JARID2 binding. When JARID2 is knocked down, there is less of the Histone 3 Lysine 27 trimethylation repressive modification at promoters. Supplementary Figure 8 supports the hypothesis of JARID2 being bound to PRC2 as one complex, and not one binding before the other. It shows that JARID2 must be associated with PRC2 before it can bind to a promoter. Supplementary Figure 9 provides further support of the ES differentiation hypothesis. Without JARID2, the ES do not differentiate properly after prolonged proliferation. Several different differentiation genes were observed and they were deregulated with the knockdown of JARID2 in ES cells. Supplementary Figure 10 explains that there is a difference between JARID2 and JARID1 family members. JARID1 and 2 have different amino acid combinations and different DNA binding domains that are not conserved. Supplementary Figure 11 builds on supplementary figure 10 by taking a closer look at the DNA binding domain of JARID2. Results conclude that the DNA binding domain of JARID2 is conserved across species.

Discussion

All of this is critical in showing that the JARID2 protein is essential in recruiting the Polycomb Repressive Complex 2 to the promoter. Without the JARID2 protein, the Polycomb Repressive Complex 2 complex does not function and therefore does not have the ability to repress DNA transcription. The Polycomb Repressive Complex 2 functions as one unit with JARID2 associated with it, as the paper has shown that JARID2 does not posses an ability to repress transcription on its own, but requires that the Polycomb Repressive Complex 2 be associated with it before it can bind and begin to methylate the histones. This is an important fact, because in mammalian cells, it is largely unknown what protein recruits the Polycomb Repressive Complex 2 to the DNA because the Polycomb Repressive Complex 2 does not have any DNA binding motifs. JARID2 has an important domain called the ARID domain, which is essential for the JARID2 protein to bind to the target gene promoter, and when mutated this protein no longer binds to the DNA promoter and components of the PRC no longer bind the promoter. This ARID domain might prove to be very important in understanding the mechanism behind JARID2 DNA binding, and therefore Polycomb Repressive Complex 2 recruitment to gene promoters.

Further studies would include using this knockout method in an animal rather than just cells, or a cancerous cell. This would be a useful technique in determining whether or not knocking down JARID2 can affect cancer cells and their ability to proliferate not only in vitro, but in vivo as well. There are many ways a cell can become cancerous, and it would be helpful to know how genes are epigenetically controlled in cancer cells so that new ways of controlling these cancerous cells can be developed. Epigenetic gene regulation through post translational modification of histones is becoming a widely studied topic because of its many uses through medical therapy.