Introduction.
Jumonji C domain-containing protein 6 (Jmjd6) belongs to a superfamily of oxygenases which rely on the presence of non-haem iron (Fe(II)) and 2-oxogluatarate for their function. The Jmjd6 gene was initially believed to be involved in engulfment of apoptotic cells as it was initially thought to encode a phosphatidylserine receptor however this is now considered unlikely. The most likely role of Jmjd6 is now believed to carry out hydroxylase and demethylase reaction and have been shown to be involved in the regulation of transcription via processes such as alternative splicing and transcriptional pause release (Böttger et al 2015). Recent research has also found a link between jmjd6 expression and the level of replication of vaccinia viruses in infected cells. This displays a potential link between the success of viral replication and the expression of jmjd6 potentially down to its transcriptional regulation function (Kwok et al, unpublished, DATE). This essay will discuss the potential functions and mechanisms of jmjd6 and then discuss these in relation to vaccinia virus infection and replication. (REDO)
Jumonji C domain-containing Protein 6-
(INTRO to JMJD6)-
First believed to be apop, then nuclear localisation etc.
Structure-
(INSERT FIG OF STRUCTURE)
The structure and domains of jmjd6 play a key role in the function and enzymatic activity the enzyme like in many other proteins it has been found to contain domains related to DNA binding as well as demethylation (reference).
AT hook-
The AT hook is a DNA binding region that is made up of a tripeptide core which is surrounded on both sides by basic amino acids. The tripeptide core was initially believed to be have to contain a glycine-arginine-proline (G-R-P) motif for DNA binding however recent studies have suggested a less stringent definition for the sequence of the AT-hook motif (Filarsky et.al, 2015). This is mostly down to the discovery of non-canonical AT-hook-like sequences which effectively bind DNA but don’t perfectly match the consensus sequence for the AT hook domain (Cikala et.al, 2004). DNA binding via the AT-hook occurs at on the minor groove of DNA. This binding alters the structure of the DNA allowing other proteins to bind to the major groove (Huth et.al, 1997). A wide range of AT- hook proteins have now been discovered. Most of these proteins have been found to have roles in the regulation of chromatin which involves processes such as histone modifications (Filarsky et.al, 2015). The presence of a non-canonical AT-hook-like sequence therefore further supports the potential roles of Jmjd6 in the regulation of transcription processes as DNA binding can occur and most other proteins that contain AT-hook domains are involved in chromatin regulation which is a key process in transcription (reference).
JmjC domain-
The JmjC domain is key for the dioxygenase activities of jmjd6. The JmjC domain has been found to have a characteristic topology via studies into the protein structure of JmjC containing proteins. This topology is the formation of a double stranded β-helix fold normally made up of eight antiparallel β-strands (Clifton et.al, 2006). This β-helix fold has the ability to form a pocket which has enzymatic activity via coordination of iron (Fe(II)) and 2-oxoglutarate. In this pocket the domain is able to carry out the oxidation (the loss) of two electrons from its main substrate. This reaction occurs at the same time where 2OG is converted into succinate and carbon dioxide (Schofield & Zhang, 1999). This is why the JmjC family of enzymes are referred to as nonheme-Fe(II)-2-oxoglutarate-dependent dioxygenases (Hahn et.al 2008). (MAYBE ADD A BIT MORE ON demeth and oxidation)
Polyserine region-
The polyserine region of Jmjd6 is made up of 16 serine residues which are split up by the presence of four aspartate amino acid residues. A study carried out by Wolf et.al (2013) found that this region was extremely important in the formation of oligomers of Jmjd6 and Jmjd6 nuclear/nucleolar shuttling. The formation of oligomers may be important in the interaction of Jmjd6 with RNA. This is suggested by the similarities between the homo-oligomeric rings formed by Jmjd6 and protein complexes that interact with RNA such as the SM ring which has been associated with pre-mRNA splicing (Meister, Eggert & Fischer, 2002). The role of the polyserine region in nuclear/nucleolar shuttling may have a key role in the transcriptional regulation function of jmjd6. This was illustrated by the jmjd6 without the polyserine region accumulating in the nucleoli instead of the nucleoplasm where the normal jmjd6 enzyme was observed to accumulate (Wolf et.al 2013). This suggest that the polyserine domain is important in allowing localisation of jmjd6 in the nucleoplasm where the activation of transcription and splicing occurs. This links to the potential role of jmjd6 in the regulation of transcription (REFERENCE).
Reactions-
Demethylation
Oxidation
(Maybe not include as there isn’t much references)
Transcription regulation-
Recent studies have suggested that the demethylation and oxidation functions of the Jmjd6 enzyme play a role in the regulation of transcription within cells. Three processes have been found to be linked to the function of Jmjd6, these are alternative splicing (Webby et.al, 2009), RNA polymerase pause release (Liu et.al, 2013) and histone arginine modification (Chang et.al, 2007). (MAYBE LINK TRANSCRIPTION REG TO VIRUS REP)
Alternative splicing-
Alternative splicing is the process by which a large number of different mRNAs and proteins are produced from a comparably low number of genes, this occurs in most metazoans. It is believed that around 60% of human genes undergo alternative splicing and many genes have multiple transcripts produced via splicing, some genes have thousands of different transcripts that are able to be produced by this process. The production of these altered mRNAs and proteins can occur due to multiple different changes to the mRNA. One of the main alterations is the inclusion or exclusion of cassette exons. These are exons are not constitutive and are only sometimes included in the transcript. The length of exons can also be altered by changes of the location of one of the genes splice sites. These changes in splice site location can have many different effects on the protein produced from the transcript such as enzymatic activity and localisation within the cell. The process of splicing is directed by splice site sequences which are observed at the junction between the intron and exon. These splice sites are found at both the 5’ end and 3’ end of introns and both contain consensus sequences to which the spliceosome can attach. The spliceosome is a large macromolecular complex that assembles around the splice site from five small nuclear ribonuleoproteins as well as multiple accessory proteins Splicing occurs through a reaction which contains two transesterification steps which are catalysed by the spliceosome. (maybe insert more about mechanism) (need references)
Research by Webby et.al (2009) using liquid chromatography-mass spectrometry techniques found a link between Jmjd6 function and the RNA splicing mechanism. Jmjd6 was observed to hydroxylate lysine residues on a key splicing regulatory factor in humans called U2AF65. It was found that Jmjd6 hydroxylated two specific lysine residues which as Lys15 and Lys276. These findings suggest a role in the regulation of alternative splicing by jmjd6.
RNA polymerase pause release-
RNA polymerase II pausing occurs during early transcriptional elongation after the enzyme has bound the RNA strand. At this point further signals are required for elongation to continue. Until these signals are released the RNA polymerase remains stably bound to the RNA. After the signals are released the polymerase then progresses along the gene, terminates and eventually restarts transcription. The process of polymerase II pausing is prevalent in metazoans and has been suggested as a widespread mechanism for transcription regulation. (MAYBE INSERT MECHANISM)
Polymerase II pausing has been observed to be common at genes which play important roles in the developmental and environmental response pathways. There are four main functions of Pol II pausing which have been suggested. Three of these are discussed below. (REFERENCE).
The role of Pol II pausing in Establishing Permissive Chromatin-
An obstacle to transcription can occur via the wrapping of promoter sequences around histones to form nucleosomes at therefore stopping important sequences to be accessible. Due to this the nucleosomes near to promoter sequences often are required to be removed. This allows the transcription machinery to access the promoter sequence and initiate transcription. This process has been displayed to be linked with gene activation in many genes. However in genes that Pol II pausing occurs this removal of nucleosomes occurs independent of whether the gene has become activated or not. These genes where Pol II has paused have been observed to be maintained in this state where the gene is accessible to regulatory-factors. This state has been found to be reliant on the presence of the Pol II pausing. This mechanism has been suggested in multiple roles such as priming genes which are constantly active at basal levels for bursts of increased transcription as a result of gene specific cues (REFERENCES).
Pol II pausing role in creating a pausing framework for quick and simultaneous gene activation-
Pol II pausing on the gene produces a strand with a promoter which can be quickly bound top by activators for the gene. This allows the gene to be quickly switched on due to the presence of paused Pol II. This has been observed in many genes present in mammals which have very fast and short lasting expressions e.g TNF-α. It has also been observed that pausing plays a role in signal transduction network. It was found that in mouse embryonic stem cells Pol II pausing was higher at promoters for genes that encode proteins involved in the signal transduction pathways. This illustrates that polII pausing is not only involved in the priming genes for activation but also been shown to have a role in in the expression of signalling molecules and transcription factors.
Pol II pausing role in Integration of regulatory signals-
Pol II pausing functions as an extra mode of regulation in transcription after polymerase binding. This could enable activators related to pause release to function along with factors involved in recruitment to control levels of transcription. Activators can have multiple effects such as general transcription factors to activate transcription or cause movement of P-TEFB to the promoter region. Therefore the combination of activators which bind is important in the levels of Pol II being recruited to the strand as well as releasing the pol II from pause. This results in a rate limiting step in transcription activation. Through this process transcription is controlled via a combinatorial control via the interactions of factors present at the promoter and enhancer regions of the gene.
The role of Pol II pausing in the checkpoints of coupling elongation and RNA processing-
(IF HAVE SPACE)
The role of Jmjd6 in RNA pol II pause release-
Jmjd6 was found to be involved in promoter-proximal pause release via co-binding distal enhancers called anti-pause enhancer. This process was linked to the promoter proximal pause release of a large group of transcription units. The release from pol II pause release is mainly down to the function of P-TEFb complex which is known to phosphorylate three targets. In cells at ~50% of the P-TEFb complex is bound to the inhibitory factor HEXIM1/2 and 7sk snRNA the other ~50% is bound to Brd4. Brd4 is able to directly release P-TEFb from the inhibitory factors (HEXIM1/2 and 7sk snRNA) via interactions with cyclin T1 which results in the P-TEFb becoming active and the phosphorylation of RNA pol II and therefore starting transcription. Jmjd6 was found to work with Brd4 as a partner in the regulation of transcription for a large subset of genes. This regulation was observed to be via their interactions with distal enhancers called anti-pause enhancers (A-PE’s). Jmjd6 was found to display demethylase activities towards the H4R3me2(s) and the 7SK snRNA cap resulting in the release of the inhibitory complex called 7SK snRNA/HEXIM. (maybe use section from initial review to discuss) (reference)
Histone arginine demethylation-
Early in the 1960s it was first observed that histones undergo post translational modification and in 1997 using the high resolution x-ray images of the nucleosomes were used to hypothesise the effect of histone modification on chromatin structure. It was observed that histone n-terminal tails can stick out from the nucleosome and touch other nearby nucleosomes. The interactions between nucleosomes were then thought to be able to be modified by post translational modifications such as demethylation. It is now known that this is true and post translational modification of histones have been displayed to play roles in recruitment of enzymes such as remodelling enzymes which remove histones using energy attained from ATP hydrolysis. There are two main mechanisms that histone modifications produce effects. The first of these is the post translational modification resulting in an alteration of the structure of chromatin. The second is the modification resulting in the positive or negative regulation of effector molecule binding. These modifications have been found to be important in transcription as well as DNA replication.
The role of Jmjd6 as a histone arginine demethylase-
In a study by Chang et.al (2007) Jmjd6 was observed to have demethylase activities against two arginine residues present on histones. These residues were the histone H3 at arginine residue 2 (H3R3me2(a)) and the histone H4 at arginine 3 (H4R3me2(a)). The methylated arginine at residue 2 on histone 4 has been found to be involved in transcription activation. This suggests that jmjd6 has a role in repression of groups of genes via its demethylation activities and hints at a role for Jmjd6 in epigenetic regulation of genes.
Vaccinia virus-
Vaccinia virus belongs to the poxviridae family of viruses which also contains other viruses such as cowpox virus and the now eradicated smallpox virus. Vaccinia virus is one of the most highly researched poxvirus partly due to its use in the production of smallpox vaccines. In the past vaccination with vaccinia virus has been linked with a number of symptoms due to side effects which include clinical symptoms such as rash formation, encephalitis and in extreme cases death. (Reference) However not all clinical symptoms occur due to vaccination with vaccinia virus. Other infections have been observed via zoonotic transmission between cattle and humans who are in frequent contact with these cattle such as dairy farmers in Brazil. These infections cause lesions on the hands and arms of infected individuals. It has now been suggested to be endemic as cases have occurred over the entire South-east of Brazil. (reference) (MAYBE REDO THIS SECTION) Human cell types that vaccinia virus mainly infect and replicate include epithelial keratinocytes and fibroblasts which are found in the airways of mammals as well as the skin (Byrd et.al, 2014).
Lifecycle-
(Insert figure)
The lifecycle of vaccinia virus produces to distinct virions with different properties and functions. The lifecycle of vaccinia virus begins with the attachment of virions to susceptible cells and entry. The method of entry depends on the type of virion attached. The two types are intracellular mature virus (IMV) and cell-associated enveloped virus (CEV) or extracellular enveloped virus (EEV) when released from the cell surface. The different methods required for cell entry is due to the different number of membranes of the two different virions. The IMV virions are surrounded by one membrane whereas the CEV/EEV virions have two membranes. These membranes have been found to be composed of different viral proteins displaying that the two virions have distinct functions as well as antigenicity. The CEV/EEV virion form has been suggested to be important in long range dissemination of the virus in the infected host whereas IMV virions are released during cell lysis and are important in more local dissemination. The reason that CEV/EEV are more suited to long range dissemination is due to being enveloped in a host cell membrane allowing evasion of the host immune system. (REFERENCE)
Viral Entry-
IMV-
The entry of IMV into cells has been displayed to involve the binding of surface proteins H3, A27 and D8 with host cell surface glucosaminoglycans or alternatively through the association of p4c with host cell laminins. After binding the virion then enters the host cell. There has been two mechanisms of entry found through research. The first is that membrane fusion of the virion and the host cell occurs independent on the pH resulting in the viral core to enter the host cell cytoplasm. The second mechanism observed was the uptake of the IMV by endocytosis after which the membrane of the virus and the endosomal membrane fuse and allow the release of the viral core into the cytoplasm. The selection of entry mechanism has been found to be related to the viral strain and the cell type of the host cell. (reference)
EEV-
Research into the entry mechanism of the EEV has been extremely difficult due to the low levels that EEV are produced and the fragile nature of virions membrane. However it has been observed using electron microscopy that the outer membrane is disrupted outside the host cell allowing the inner membrane to fuse with the host cells plasma membrane and entry of the viral core.
Viral gene expression-
Viral entry mechanisms allow the viral core to enter the cells cytoplasm. This core contains the viral DNA along with viral transcription enzymes. The core is them carried further into the cytoplasm on microtubules. The interactions between the microtubules and the core that allow this transport are currently unidentified. The microtubules carry the cores to the perinuclear region of the cytoplasm where they become partially uncoated allowing the viral DNA to be transcribed into messenger RNA. This transcription is carried out by DNA-dependant RNA polymerase which is one of the transcription enzymes present in the viral core.
The viral genome is divided into early and late genes. Around 50% of the viral genome are transcribed early in infection. The early genes are divided further into early and intermediately expressed genes. The early genes produce proteins involved in viral DNA replication and evasion of innate cell immune whereas intermediate genes are activated after transcription and mainly encode proteins that activate the late genes. These late genes produce the proteins involved in making the virions and enzymes required in the viral core.
Virion Production-
Virion production occurs in areas of the cytoplasm which lack organelles called viral factories. An oval spherical structure is observed to grow first consisting of proteins and lipids this surrounds the viral core components. This structure consisting of the protein lipid coat and the viral core components is called an immature virion. Viral DNA is then packed into this immature virion and cleavage of viral core proteins results in the production of IMV virions. Most virions remain at this stage and remain in the cell until cell lysis. The rest of the virions are then moved from the viral factories to areas of wrapping such as the trans-golgi network where they are wrapped by a double membrane derived from the host. These virions are called intracellular enveloped virus (IEV) which are then transported to the outer edge of the cell along microtubules. The movement of these virions have been found to be dependent on F12 protein which is a vaccinia virus protein. Deletion of F12 ligand has been shown to have a dramatic effect on the levels of CEV formation at the cell surface.
EEV release-
For EEV virions to be released the IEV virions must successfully pass through a layer of cortical actin to reach the host cells plasma membrane. This is believed to be achieved through vaccinia virus modifying the cells peripheral microtubules and actin filaments through the inhibition of RhoA signalling via the vaccinia virus protein F11. When the IEV virion has crossed the layer of cortical actin the outer membrane of the IEV integrates with the plasma membrane resulting in the enveloped virus being positioned onto the cell surface. During this integration a protein called A36 begins to build up in increasing levels below the CEV. The protein is then phosphorylated by src kinases causing the virion to disassociate from the microtubules and a signalling cascade is activated resulting in actin polymerisation below the CEV virion. This results in the formation of actin tails which push the virion outwards, away from the host cell resulting in the formation of an EEV virion.
Vaccinia virus Dissemination-
The dissemination of vaccinia virus in the host is believed to be mostly down to the release of EEV. This is due to the fact that EEVs are covered in a host derived membrane that gives them protection against neutralising host antibodies whereas IMV virions have been found to be susceptible to host neutralising antibodies as well as the complement system. This has been supported further by research such as the observation that virus strains that produce IMV virions only have been found shown to be avirulent and display an inability to spread in vivo.
The potential role of macrophages in Vaccinia virus replication and dissemination-
The mechanism of long range vaccinia virus dissemination in human are unknown however a recent study by Byrd et.al (2014) suggest a role of macrophages in this process. Vaccinia virus can produce a generalised viral infection of the host like that of smallpox but much less severe. During this vaccinia viremia skin lesions occur on the host. Studies of these skin lesions in vaccinia virus infected murine models displayed the presence of mobile macrophages infected with vaccinia virus next to the infected skin cells. Analysis of these macrophages displayed that these cells allowed permissive vaccinia virus infections and contained 7% of the vaccinia virus concentration in the lesion (Hickman, et.al, 2013). However it has been displayed that vaccinia virus infections in primary human monocytes produce an abortive infection where no virus replication occurs (Broder et.al, 1994). In the study by Byrd et.al (2014) it was observed that M1-polarised and M2-polarised macrophages produces ~15.3 and ~31.7 plaque forming units/cell 48 hours after infection with vaccinia virus at a multiplicity of infection of 5. This suggests that in vaccinia infections macrophages may carry a significant proportion of the viral burden. The difference in viral load between the two macrophage types may be down to their function. M1 macrophages are known to perform a pro-inflammatory role in the host’s immune system producing antiviral compounds, whereas M2 macrophages are known to have anti-inflammatory functions. The production of antiviral compounds by activated M1 macrophages may be the cause of the lower levels of vaccinia virus present in this cell type due to intracellular viral killing. It was also observed that infected macrophages produced mainly EEV virions inconsistent with the levels of virion production in other cell types where IMV virions are the most prominent. Overall this displays a possible role of macrophages in the further dissemination of vaccinia virus through the host due to the movement of macrophages in the circulation and lymph systems as well the majority of virions being produced being EEV which have been observed to be linked to long range dissemination due to being enveloped in a host derived membrane. (REFERENCES)
Jmjd6 role in vaccinia virus replication-
The role of Jmjd6 in vaccinia virus replication has been recently studied by Kwok et.al (unpublished). In this work it was observed that EEV vaccinia virion release from HeLa cells was reduced by almost tenfold in cells in which siRNA-275 was used to knockdown jmjd6 expression wheras cell associated IMV and CEV virions were unaffected. This suggests that jmjd6 may have a role in the effective production of EEV virions by vaccinia virus. This reduction may be down to the role that Jmjd6 has been observed to play in cell transcription regulation. If genes required for effective release of CEV virions is regulated by Jmjd6 the knockdown of this gene may explain the reduction in EEV release. In this study it was also observed using fluorescence microscopy techniques that jmjd6 knocked down cells displayed abnormal viral factory morphology. The viral factories were observed to be form further away from the nucleus rather than in normal vaccinia virus infections where they are situated close to the nucleus in the perinuclear space. This may be due to the role of jmjd6 in transcription regulation processes such as histone modification which could result in genes essential for the effective transport to the perinuclear space to be expressed at lower levels or not at all. This would therefore mean viral factories forming further from the perinuclear space as observed.
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