JMJD6 cleaves MePCE to release positive transcription elongation factor b (P-TEFb) in higher eukaryotes
Mechanisms of transcription regulation in bacteria are very well established; transcription factors bind to specific DNA to recruit RNA Polymerases ( RNAP ) to carry out transcription ( Ptashne and Gann , 1997; Zhang et al . , 1999 ) . In eukaryotes , however , there are additional layers of regulation such as the nucleosome structures , which can prevent RNA Polymerases including RNA Polymerase I ( Pol I ) , RNA Polymerase II ( Pol II ) , and RNA Polymerase III ( Pol III ) from productive transcription due to high binding affinity between DNA and histones . Precisely how RNA Polymerases overcome nucleosomal barriers to undergo a productive transcription elongation and how Pol II pausing is regulated remain unanswered ( Zhou et al . , 2012; Jonkers and Lis , 2015; Core and Adelman , 2019 ) . In higher eukaryotes , over ~30% genes are regulated by Pol II promoter-proximal pausing ( Core et al . , 2008; Nechaev et al . , 2010; Min et al . , 2011 ) , which is resultant from nucleosome barriers at +1 position of transcription start sites ( Weber et al . , 2014; Voong et al . , 2016 ) . We recently discovered that a group of JmjC domain containing protein family including JMJD5 and JMJD7 specifically cleave histone tails and potentially generate tailless nucleosomes . The cleavage activity by JMJD5 and JMJD7 could be associated with the release of the promoter-proximal paused Pol II and may trigger Pol II into productive elongation in higher eukaryotes , such as mouse and human ( Liu et al . , 2017; Liu et al . , 2018 ) . The cleavage activity of JMJD5 on histone tails was also independently reported by another group ( Shen et al . , 2017 ) , thus cross-validating our respective discoveries . Compared to the efficient recruitment of P-TEFb ( including CDK9 and Cyclin T1 ) by TAT protein in human immunodeficiency virus ( HIV ) ( Peterlin et al . , 2012 ) , BRD4 is claimed to be responsible for the recruitment P-TEFb to the promoters of Pol II pausing regulated genes ( Jang et al . , 2005; Yang et al . , 2005; C Quaresma et al . , 2016 ) . However , the binding affinity between BRD4 and P-TEFb ( ~0 . 5 μM ) ( Itzen et al . , 2014 ) is much weaker than that that of TAT and P-TEFb ( ~3 nM ) ( Wei et al . , 1998; Tahirov et al . , 2010; Schulze-Gahmen et al . , 2014 ) , and BRD4 lacks a RNA binding motif ( Wu and Chiang , 2007 ) . Therefore , we hypothesize there must exist another factor to help BRD4 to recruit P-TEFb and engages in the instigation of Pol II transcription elongation . Besides the classic Bromo-domains which recognize acetylated histone tails , BRD4 contains an extra terminal domain ( ET ) recognizing JMJD6 ( Rahman et al . , 2011; Konuma et al . , 2017 ) . Incidentally , we found that JMJD6 nonspecifically binds to single stranded RNA with high affinity ( ~40 nM ) ( Hong et al . , 2010 ) . We propose that JMJD6 may be recruited by both BRD4 and newly transcribed RNAs from Pol II to help BRD4 recruit P-TEFb , acting analogously to that of TAT protein associating with both P-TEFb and TAR . JMJD6 is one of the most controversial proteins in biology ( Vangimalla et al . , 2017 ) . It was first cloned as phosphatidylserine ( PS ) receptor ( Fadok et al . , 2000 ) , but was corrected as a nucleus expressed protein unrelated to PS ( Böse et al . , 2004; Cikala et al . , 2004; Cui et al . , 2004 ) . It was later reported to contain arginine demethylase activity on histone tails ( Chang et al . , 2007 ) , hydroxylase activity on splicing factor U2AF65 ( Webby et al . , 2009 ) and histone tails ( Han et al . , 2012 ) , and both arginine demethylase activities on histone tails and RNA demethylase activities on 5’ prime of 7SK snRNA ( Liu et al . , 2013 ) , and surprisingly PS binding ( Neumann et al . , 2015; Yang et al . , 2015 ) . The exact or cognate substrate ( s ) of JMJD6 remains unresolved or controversial . Based on the novel protease activities of JMJD5 and JMJD7 ( Liu et al . , 2017; Shen et al . , 2017; Liu et al . , 2018 ) , the high structural similarity among catalytic cores of JMJD5 , JMJD6 , and JMJD7 ( Hong et al . , 2010; Liu et al . , 2018 ) , and analogous severe phenotypes among knockouts of Jmjd5 and Jmjd6 in mice ( Li et al . , 2003; Böse et al . , 2004; Ishimura et al . , 2012; Oh and Janknecht , 2012 ) , we hypothesized that JMJD6 may contain protease activity working on methylated arginines on some protein candidates which regulate the activity of Pol II , especially promoter-proximally paused Pol II . It is well established that the 7SK snRNP complex primarily functions to sequester the CDK9-containing P-TEFb until stimulation ( Jang et al . , 2005; Yang et al . , 2005 ) . MePCE ( methylphosphate capping enzyme ) was first characterized as a component of the 7SK snRNP complex which acts as a capping enzyme on the gamma phosphate at the 5’end of 7SK RNA ( Jeronimo et al . , 2007 ) . Furthermore , a capping-independent function of MePCE via stabilization of 7SK snRNA and facilitation in the assembly of 7SK snRNP was reported by Dr . Qiang Zhou’s group ( Xue et al . , 2010 ) . Knockdown of MePCE led to destabilization of the 7SK snRNP complex in vivo ( Xue et al . , 2010; Singh et al . , 2011; C Quaresma et al . , 2016 ) . A nonsense variant of MePCE is reported to be associated with a neurodevelopmental disorder exhibiting hyperphosphorylation of Pol II , potentially caused by enhanced activation of CDK9 complex ( Schneeberger et al . , 2019 ) . Interestingly , one report showed that MePCE may also work in an 7SK snRNP independent manner to recruit CDK9 on a small group of genes ( Shelton et al . , 2018 ) . In this report , we reveal that MePCE of the 7SK snRNP complex is a cognate substrate of JMJD6 .
Based on these divergent reports regarding substrates of JMJD6 ( Chang et al . , 2007; Webby et al . , 2009; Han et al . , 2012; Liu et al . , 2013; Neumann et al . , 2015 ) , we re-interrogated proposed substrates using stringent and unified criteria . As we reported previously , JMJD6 binds with high binding affinity ( ~40 nM ) to single stranded RNA ( ssRNA ) without sequence specificity ( Hong et al . , 2010 ) . However , truncation analysis showed that JMJD6 barely binds to ssRNA without the C-terminal flexible region ( Hong et al . , 2010 ) . This suggests that the C-terminal domain of JMJD6 may just serve as ssRNA binding motif and RNAs are not a substrate for the enzymatic activity of JMJD6 . On the other hand , the structure of the catalytic core of JMJD6 shows some critical similarity to those of JMJD5 and JMJD7 , with a negatively charged microenvironment near the catalytic center ( Hong et al . , 2010; Liu et al . , 2018 ) , suggesting positively charged substrates ( Figure 1 ) . As we reported , JMJD5 and JMJD7 specifically recognize methylarginines of histone tails via a Tudor-domain-like structure near the catalytic center of JMJD5 , which could specifically recognize methylarginines , but not methyllysine ( Liu et al . , 2017; Liu et al . , 2018 ) . We reasoned that the similar structural features among JMJD6 , JMJD5 , and JMJD7 may confer a similar substrate for JMJD6 as those of JMJD5 and JMJD7 . In this regard , crystals of JMJD6 without C-terminal motif ( 1-343 ) were soaked with a monomethylarginine derivative . Interestingly , four out of eight JMJD6 molecules within an asymmetric unit bound to monomethylarginine , which coordinates with Fe2+ and alpha-KG in the catalytic center similar to that of JMJD5 and methylarginines ( Figure 1 , Figure 1—figure supplements 1–3 , Supplementary file 1 ) . However , the methylated sidechain of arginine is located in a more open catalytic space containing negatively charged residues , compared to that of JMJD5 , indicating that the pocket could hold more than one sidechain ( Figure 1A ) . This may suggest a novel substrate recognition mode , which is different from that of JMJD5 ( Liu et al . , 2018 ) . Nevertheless , the complex structure shows several key evidences . First , JMJD6 does bind to substrates with methylarginine or possibly methyllysine or both arginine and lysine with and without methylation ( Figure 1B , C ) . Second , the methyl group is far away from either the divalent ion or alpha-KG , suggesting JMJD6 may not act as lysine or arginine demethylases to remove methyl groups on the sidechain of either lysine or arginine . Third , peptides or proteins could be cognate substrates instead of RNA or DNA , which do not contain any positive charge with or without methylation . Fourth , the catalytic center contains analogous residues present in JMJD5 and JMJD7 , suggesting a similar novel catalytic mechanism as those of JMJD5 and JMJD7 through an imidic acid as proton mediator ( Figure 1D; Lee et al . , 2017; Liu et al . , 2018 ) . This novelty is particularly exemplified by the fact that commercially available protease inhibitor cocktail ( Roche ) at 2x concentration cannot inhibit activities of JMJD5 , JMJD6 , and JMJD7 . Furthermore , a comprehensive protein composition analysis using mass spectrometry ( Supplementary file 2 ) of all purified recombinant JMJD6 samples used in the following experiments could not detect any protease contaminants . Since JMJD5 and JMJD7 make cleavage on histone tails ( Liu et al . , 2017 ) , we asked whether JMJD6 also recognizes histone tails . Interestingly , JMJD6 does in fact have activities on bulk histone in vitro ( Figure 2—figure supplement 1A ) . This result may explain why two groups found that JMJD6 could reduce methylarginine containing histone tails in in vitro assays probed with antibodies ( Chang et al . , 2007; Liu et al . , 2013 ) , in which post-cleft short peptides with methylarginines cannot be recognized by antibodies in western blots . To confirm or rule out whether histone tails are cognate substrates of JMJD6 , we first assessed the binding affinity between JMJD6 and histone peptides with or without arginine methylation . From fluorescence polarization binding assays , binding affinities between JMJD6 and peptides are weak and around ~150 μM ( Figure 3—figure supplement 1D , E ) . Most importantly , with or without Jmjd6 , the level of arginine methylated histones or overall histone levels does not change in MEF cells in vivo ( Figure 2—figure supplement 1B ) , which is in stark contrast to those of JMJD5 and JMJD7 ( Liu et al . , 2017 ) . These data suggest that histone tails are not cognate substrates of JMJD6 . Parenthetically , it is reported that JMJD6 binds to LARP7 on 7SK snRNP ( Weimann et al . , 2013 ) , works on methyl cap of 5’ 7SK snRNA ( Liu et al . , 2013 ) , and binds to BRD4 ( Rahman et al . , 2011; Konuma et al . , 2017 ) , all suggesting a close relation with the 7SK snRNP complex . We hypothesized that JMJD6 may work on some protein component ( s ) of the 7SK snRNP to regulate the stability of 7SK snRNP complex . Interestingly , all protein components of the 7SK snRNP complex including MEPCE , LARP7 , and HEXIM1 are drastically decreased in the MEF cells without Jmjd6 ( Figure 2A ) . One possibility is that all members are transcriptionally regulated by JMJD6 . This could be consistent with the severe phenotype of Jmjd6 knockouts , which results in neonatal lethality and cell growth retardation and tissue differentiation ( Li et al . , 2003; Böse et al . , 2004 ) , suggesting that JMJD6 is a global master transcriptional regulator controlling expression of a large group of genes including components from the 7SK snRNP complex . However , our RNA-Seq data does not support direct transcriptional downregulation of 7SK snRNP complex members at mRNA level ( Supplementary file 3 ) . All of them , including MePCE , LARP7 , and HEXIM1 have similar mRNA levels with or without Jmjd6 . At the moment , the cause of this downregulation of proteins level of these components from 7SK snRNP remains unresolved and is beyond the scope of this report . However , when we introduce back JMJD6 via overexpression into MEF cells lacking Jmjd6 , protein levels of LARP7 and HEXIM1 are rescued , whereas MePCE is undetectable ( Figure 2A ) or significantly reduced ( Figure 2—figure supplement 1C ) . To account for this disappearance of MePCE , we hypothesized that overexpression of JMJD6 may directly target MEPCE for degradation . To confirm this hypothesis , we respectively overexpressed full-length MePCE in wild-type MEF , Jmjd6 knockout MEF , wild-type JMJD6 overexpression in Jmjd6 knockout MEF , and inactive mutant JMJD6 overexpression in Jmjd6 knockout MEF . The whole cell lysates were probed with anti-MePCE antibody . Our hypothesis was vindicated with the emergence of a lower molecular weight form of MePCE in wild-type MEF and wild-type JMJD6 overexpression in Jmjd6 knockout MEF , whereas the lower molecular weight form of MePCE was not detectable in the Jmjd6 knockout MEF , and inactive mutant JMJD6 overexpression in Jmjd6 knockout MEF ( Figure 2B ) . To reproduce these in vivo results in an in vitro setting , full-length proteins linked with N-terminal His6-tag of MePCE and HEXIM1 were generated from insect cells and LARP7 and HEXIM2 were generated from bacteria and purified , followed by subjecting these proteins to direct in vitro enzymatic assays . Consistent with our in vivo results , JMJD6 cleaves MePCE in vitro ( Figure 2C , D ) , but not HEXIM1 , HEXIM2 , nor LARP7 ( data not shown ) . Upon cleavage of MePCE by JMJD6 in vitro , a band with molecular weight ~25 kDa was detected by antibody against His6-tag at the N-terminal ( Figure 2C ) . To provide context , MePCE contains a total of 689 residues and a theoretical molecular weight of 74 . 4 kDa , whereas it is detected at ~90 kDa on SDS-PAGE gel due to its high content of proline residues ( 10 . 7% ) . We expect that the ~25 kDa fragment from N-terminal of MePCE may contain 200 residues or less due to rich proline residues within the N-terminal of MePCE ( first 150 residues of MePCE contains 27 prolines , 18 . 0% prolines ) . Furthermore , our in vivo assay yielded a cleaved MePCE band with a molecular weight ~65 kDa ( Figure 2B ) . The anti-MePCE antibody used in this assay was generated using a peptide immunogen containing residues 200–250 of MePCE . Thus , the ~65 kDa band may correspond to the cleaved C-terminal segment of MePCE containing the approximate residues 200–689 . Based on the binding of JMJD6 to methylated arginine we obtained from the complex crystal structure , we reasoned that arginine residues within approximately the first 200 residues of MePCE could contain the recognition site . Several peptide fragments including residues from 81 to 160 , residues 154 to 184 , and residues 187 to 244 were synthesized . Peptides of 81–160 and 187–244 did not show any cleavage when incubated with JMJD6 . Peptides of 154–184 showed cleavage activity , but at levels of <1% compared to peptide input . We attributed this low activity as a matter of insolubility and dimerization via C177 oxidation . To overcome this obstacle , a shorter peptide from residue 161 to 179 containing C177S was synthesized and subjected to enzymatic reaction under JMJD6 . This peptide also contains symmetric dimethylation on R171 , given that our binding data , described in the next section , suggests that this particular modification yields the highest binding affinity ( Figure 3C ) . In line with our expectations , dominant peaks corresponding to the cleaved MePCE peptide products were detected by mass spectrometry , but not in either peptide alone or with an inactive mutant version of JMJD6 ( Figure 2E ) . To determine the binding affinity between JMJD6 and the newly discerned MePCE proteolysis site , Microscale Thermophoresis ( MST ) assay was performed using catalytic core of JMJD6 ( 1–343 ) titrated with the MePCE ( 154-184 ) peptide exhibiting: no modification ( Figure 3A ) , R170-me2s ( Figure 3B ) , or R171-me2s ( Figure 3C ) . The highest binding affinity was exhibited by the MePCE ( 154-184 ) -R171-me2s peptide with Kd = 159 ± 77 nM , followed by the peptide containing no modification with Kd = 471 ± 292 nM , and lastly the peptide with R170-me2s with Kd = 568 ± 316 nM . Fluorescence polarization assays were used to cross-validate the Kd values obtained via MST above , all of which respectively fell within the margin of error ( Figure 3—figure supplement 1A , B , C ) . These data suggest that methylation on R171 enhances binding nearly 3-fold compared to no modification , whereas methylation on R170 diminishes binding marginally . Whether or not these particular methylations occur in vivo is yet to be explored . Interestingly , the YASARA Energy Minimization Server developed by Dr . Kevin Karplus’ group yielded a theoretical model that positions R171 in the identical position to the monomethyl-arginine observed in our crystal model ( Figure 3—figure supplement 2A; Krieger et al . , 2009 ) . Furthermore , the energy-minimized model displayed eight separate charge-charge interactions between JMJD6 and MePCE ( Figure 3G ) . Although a bona fide complex structure is preferred , this computational model is in excellent agreement with all our findings and provides a coherent justification for the experimentally observed high binding affinities between JMJD6 and MePCE . To compare the abovementioned binding affinities to that of JMJD6 and histone tails , as other groups have previously purported , the identical MST assay was performed using peptides derived from Histone 3 ( 1-21 ) containing no modification ( H3 ) ( Figure 3D ) , Histone 3 ( 1-16 ) -R2-me2s ( H3R2me2s ) ( Figure 3E ) , and C-peptide ( 57-87 ) as a negative control ( Figure 3F ) . For the Histone three peptides , low binding affinity was exhibited with Kd = 77 ± 34 μM for H3 peptide and Kd = 98 ± 14 μM for H3R2me2s peptide . No meaningful binding was observed for the C-peptide ( Figure 3F ) . To verify the authenticity of the above model and confirm our proposed cleavage site of JMJD6 within MePCE , we generated an MePCE knockout 293 T cell line ( Figure 3H ) and respectively overexpressed C-terminal His6-tagged wild-type MePCE or C-terminal His6-tagged mutant MePCE , which replaced -RRRR- to -AAAA- at the cleavage site . Interestingly , when probed with anti-His6 antibody in a western blot , wild-type MePCE generates two bands of MePCE ( Figure 3I , left lane ) , consistent with Figure 2B , whereas the mutant version does not ( Figure 3I , right lane ) , suggesting that this site is the authentic cleavage site for JMJD6 . To further confirm these results , we introduced the two variants of MePCE back to the JMJD6 overexpressing MEF cell line in jmjd6 knockout background . Once again consistent with Figure 2B , two major bands show up with the wild-type MePCE ( Figure 3J , left lane ) , whereas only one major band and one faint band show up with the mutant MePCE ( Figure 3J , right lane ) . This data provides another solid evidence of the cleavage site within MePCE by JMJD6 . To further verify our proposed role of JMJD6 on MePCE , the mutant MePCE containing N-terminal His6-tagged was expressed in insect cells and purified for an in vitro assay . The native form of MePCE generates a small band ( Figure 3—figure supplement 2B ) , consistent with Figure 2C , whereas the mutant MePCE does not generate any additional band ( Figure 3—figure supplement 2C ) . Taken together , we propose that JMJD6 is the cognate protease of MePCE and cleaves at the R171 site within MePCE . We previously reported that cleavage of histone tails by JMJD5 and JMJD7 could lead to tailless nucleosomes , which are unstable and are more readily overcome by Pol II during transcription ( Liu et al . , 2017; Liu et al . , 2018 ) . What happens when MePCE is cleaved ? Does MePCE cleavage mediate P-TEFb to be released from the 7SK snRNP complex , allowing P-TEFb to be recruited to CTD of Pol II , and then to phosphorylate Ser2-CTD of Pol II ? Nuclear extracts from both wild-type MEFs , Jmjd6 knockout MEFs and JMJD6 overexpression in Jmjd6-deficient MEFs were subjected to investigation as to the level of Ser2p-CTD of Pol II . Consistent with all our current and previous findings , Ser2p-CTD of Pol II is drastically reduced ( ~80% ) in MEF cells without Jmjd6 , despite CDK9 levels remaining unchanged , suggesting that Ser2p-CTD of Pol II is regulated by JMJD6 ( Figure 4A , B ) . A question raised here is whether the drop of active Pol II ( Ser2p-CTD ) is related to the protease activity of JMJD6 toward the CDK9-sequestering role of MePCE . Therefore , the MePCE knockout cell line was investigated for its effect on Pol II . To our great satisfaction , Pol II Ser2p-CTD is dramatically increased in 293 T cells with MePCE knockout ( Figure 4C , right lane ) compared to the wild-type ( Figure 4C , left lane ) . Of interest , Shelton et al . ( 2018 ) showed that CDK9 interacts with the N-terminal region of MePCE ( 1-400 ) ) . We found a very similar JMJD6 cutting site around residues 363–367 ( -RKRRR- ) compared the site we characterized around residues 167–172 ( -KRRRR- ) . If JMJD6 also cuts this site as well , it seems that MePCE may act as an anchor site for CDK9 on 7SK snRNP complex . It is an interesting topic that requires further investigation . The totality of our results is consistent with the critical role of JMJD6 in embryonic development , which is neonatal lethal without Jmjd6 ( Li et al . , 2003; Böse et al . , 2004 ) . It explains why growth of MEF cells without Jmjd6 is severely compromised , similar to those without either Jmjd5 or Jmjd7 ( Liu et al . , 2017 ) . This data suggests not only that JMJD6’s cleavage of MePCE is essential for P-TEFb to be released from the 7SK snRNP complex and ultimately the phosphorylation of Ser2-CTD of Pol II , but also further cross-confirms that a species of Ser2p-CTD of Pol II is indeed generated by CDK9 . The content of Ser2p-CTD of Pol II is recovered when JMJD6 is re-expressed ( Figure 4A , B ) , suggesting the direct relation of JMJD6 and phosphorylation of Ser2-CTD of Pol II in vivo . When these results are aggregated with previously published reports , whereby ( 1 ) JMJD6 exhibits strong binding affinity to nonspecific ssRNA as we previously reported ( Figure 4D; Hong et al . , 2010 ) , ( 2 ) JMJD6 associates with BRD4 as reported by others ( Figure 4D; Rahman et al . , 2011; Konuma et al . , 2017 ) , and ( 3 ) JMJD6 digests MePCE so as to disrupt the overall stability of 7SK snRNP complex as highlighted in this report ( Figure 4D ) , a model of P-TEFb transcription regulation unique to higher eukaryotes can be generated: JMJD6 binds to 20–50 nt newly transcribed 5’ prime end of ssRNAs of initiated Pol II of any stimulating genes . The association between BRD4 and JMJD6 allows BRD4 to bring P-TEFb ( CDK9 ) to close proximity of CTD of Pol II , allowing for Ser2-CTD phosphorylation ( Figure 4E ) . Remarkably , this recruitment of P-TEFb by JMJD6 , with the assistance of BRD4 , is similar to that of TAT protein hijacking P-TEFb complex after associating with TAR transcript to trigger the expression of HIV retroviral genome ( Figure 4F; Peterlin et al . , 2012; Zhou et al . , 2012; C Quaresma et al . , 2016 ) . Here , our observation regarding the direct relation of JMJD6 and level of Ser2p-CTD of Pol II in vivo is corroborated by Liu et al . ’s report , in which 7SK snRNP complex pulled-down through HEXIM1 antibody from HEK293 cells could be disrupted by JMJD6 in vitro , as indicated by the decrease in CDK9 levels ( Liu et al . , 2013 ) , thus strongly supporting our proposed role of JMJD6 as a direct disruptor of the 7SK snRNP complex .
It took us nearly two decades to address the structure and function of JMJD6 since it was first cloned ( Fadok et al . , 2000 ) . During the long journey , based on the conserved structure of JmjC domain containing family , we pioneered in characterizing the catalytic core of JMJD2 subfamily , which turned out to be a novel lysine demethylase ( Chen et al . , 2006; Whetstine et al . , 2006; Chen et al . , 2007 ) . We then solved the structure of JMJD6 with the help of a monoclonal antibody and discovered its unique ssRNA binding property almost a decade ago ( Hong et al . , 2010 ) . However , the exact function and its cognate substrate remained a mystery , though several groups claimed variety of enzymatic activities and substrates in past two decades ( Chang et al . , 2007; Webby et al . , 2009; Han et al . , 2012; Liu et al . , 2013 ) . The accidental discoveries of novel protease activities of JMJD5 and JMJD7 by our group prompted the exploration of potential protease activity of JMJD6 ( Liu et al . , 2017; Liu et al . , 2018 ) . The report by Liu et al . ( 2013 ) helped us to narrow down the potential cognate substrate within the 7SK snRNP complex . Reports of arginine demethylase activity ( Chang et al . , 2007; Liu et al . , 2013 ) helped us to focus on methylated arginine as the potential cleavage sites . Thanks to these reports , we identified MePCE to be the cognate substrate of JMJD6 , as well as the potential cleavage site . Numerous lines of evidence from our current discoveries and other publications corroborates the authenticity of this conclusion: First , JMJD6 is specifically associated with BRD4 through the ET domain ( Rahman et al . , 2011; Konuma et al . , 2017 ) , while BRD4 is very well established to recruit P-TEFb to paused Pol II ( Yang et al . , 2005; Yang et al . , 2008 ) . Second , JMJD6 could disrupt the 7SK snRNP complex in vitro as reported by Liu et al . ( 2013 ) . Third , our current discoveries showed that JMJD6 cleaves MePCE of the 7SK snRNP complex . Fourth , our previous discovery of JMJD6’s interaction with non-specific ssRNA bridges JMJD6 to the initiated Pol II which generates 20–50 nt long ssRNA ( Hong et al . , 2010 ) . The findings that JMJD5 and JMJD7 are in fact proteases was highly unexpected , and required multiple lines of evidence pursued with great scrutiny . It is quite extraordinary that a very conserved Jumonji domain containing hydroxylase family should contain a subfamily which evolutionarily adopted distinctive protease activities , as well as exhibit several unexpected novelties including the catalysis mechanism involving imidic acid and both endopeptidase and exopeptidase activities ( Liu et al . , 2017; Liu et al . , 2018 ) . Indeed , recombinant proteins derived from bacteria have a high chance of contamination by bacterial proteases , although we exhausted innumerable means to exclude the possibility of contamination in our assays ( Liu et al . , 2017; Liu et al . , 2018 ) . In this report , the novel protease activity of JMJD6 , which cleaves before the methylated arginine , is remarkably distinct to the cleavage after the methylated arginine mediated by JMJD5 and JMJD7 ( Liu et al . , 2017; Liu et al . , 2018 ) . This important piece of evidence supports the claim of JMJD5 and JMJD7 as proteases . It is highly improbable that one batch of proteins we purified was contaminated by one type of undiscovered protease ( s ) ( cleavage after methylated arginine for JMJD5 and JMJD7 ) while another batch of proteins was contaminated by a different type of undiscovered protease ( s ) ( cleavage before methylated arginine for JMJD6 ) , all which respectively have substrate specificity matching the expected biological profiles of JMJD5/6/7 . Based on our current discoveries , we may derive a novel transcription regulation pathway for genes regulated by promoter-proximal pausing Pol II . First , upon stimulation of cells , signals will reach specific transcription factors through signal transduction pathways . With modification or with the help of other partner molecules , these transcription factors bind to enhancers close to the paused Pol II . These transcription factors will recruit P300/CBP with the help of H3K4 ( me1 ) at enhancer regions , which in turn acetylates H3 and/or H4 on the same nucleosome bound by P300/CBP through association with H3K4 ( me1 ) to generate acetylated H3 and/or H4 ( Figure 5A ) . Next , BRD4 is recruited to acetylated H3 and/or H4 , and in turn engages with JMJD6 and the initiated Pol II complex . JMJD6 specifically cleaves MePCE of the 7SK snRNP complex so as to release the P-TEFb complex containing CDK9 ( Figure 5B ) . Finally , JMJD6 and BRD4 brings P-TEFb to close proximity of CTD of Pol II through super elongation complex . P-TEFb ( CDK9 ) then phosphorylates Ser2-CTD of Pol II ( Figure 5C ) .
The cDNA corresponding to gene of wild-type JMJD6 ( 1–343 ) , inactive mutant JMJD6 ( 1–343 ) H187A/D189A/H273A/K204A/N287A , HEXIM2 , and LARP7 was cloned into a pET28a vector containing an N-terminal His6 tag . The DNA corresponding to gene of JMJD6 ( 1–403 ) wild-type was cloned into a pGEX vector containing an N-terminal GST tag and TEV linker . All proteins were expressed in Rosetta ( DE3 ) Escherichia coli cells . All cell cultures were grown to A600 value equal to about 1 . 0 and induced with a final concentration of 1 . 0 mM isopropyl 1-thio-β-D-galactopyranoside overnight at 16°C . Cells were resuspended in nickel-binding buffer ( 50 mM Tris-HCl , pH8 . 0 , 1M NaCl , 1 mM PMSF ) and lysed using a sonicator ( Fisher Scientific Sonic Dismembrator Model 500 ) at 35% power , 10 s ON , 5 s OFF , for 20 min . The lysate was centrifuged at 16 , 000 rpm at 4°C for 30 min . The supernatant corresponding to His-JMJD6 ( 1–343 ) was loaded to 7 mL of Ni-NTA resin ( GE Healthcare ) , washed with nickel-binding buffer containing 20 mM imidazole , and eluted with nickel-binding buffer containing 500 mM imidazole . The supernatant corresponding to GST-tev-JMJD6 ( 1–403 ) was loaded to 7 mL of glutathione agarose resin ( Thermo Scientific ) , washed with nickel-binding buffer , and eluted with nickel-binding buffer containing 30 mM glutathione . All eluted JMJD6 products were concentrated and purified on a superdex 200 10/300 GL column ( GE Healthcare ) previously equilibrated with nickel-binding buffer containing 15 mM β-mercaptoethanol . The cDNA fragment encoding full-length wild-type MePCE with an N-terminal His6-tag , full-length mutant MePCE ( R169D/R170N/R171D/R172N ) with an N-terminal His6-tag , and HEXIM1 with an N-terminal His6-tag was cloned under control of the polyhedrin promoter into a previously described baculovirus transfer plasmid ( Kozono et al . , 1994 ) . Recombinant virus was made by co-transfection into SF9 insect cells ( Invitrogen ) of the plasmid and BacVector3000 baculovirus DNA ( Novagen ) using the calcium phosphate co-precipitation method . High titer virus stock was prepared by infection of SF9 insect cells . Wild-type MePCE , mutant MePCE , and HEXIM1 protein was produced by infection of High Five insect cells ( Invitrogen ) at high multiplicity of infection . Four days later , the cells were lysed by sonication in nickel-binding buffer . Lysates were cleared by centrifugation ( 20 , 000 rpm , 60 min ) and proteins were purified from the supernatant using 7 mL of Ni-NTA resin ( GE Healthcare ) . The protein was eluted from the column with nickel-binding buffer containing 500 mM imidazole . His-JMJD6 ( 1–343 ) was crystallized by vapor diffusion in sitting drops with 0 . 1M sodium citrate pH 5 . 6 , 1 . 0M ammonium phosphate monobasic at 8°C . The crystals were soaked in soaking buffer; 0 . 1M sodium citrate pH 5 . 6 , 1 . 0M ammonium phosphate monobasic , 10 mM mono-methyl arginine , 3 . 75 mM αKG , 3 . 75 mM Iron ( II ) sulfate . For data collection , His-JMJD6 ( 1–343 ) crystals were transferred to a cryo-protecting buffer ( soaking buffer supplemented with 25% glycerol ( v/v ) ) and frozen in liquid nitrogen . All data used in structure solving and refinement were collected on a beam line 4 . 2 . 2 ( MBC-ALS ) at the Advanced Light Source ( Berkeley , ALS , USA ) . Data were integrated and scaled using the HKL2000 suite of programs . Structural determination and refinement results are shown in Supplementary file 1 . To analyze protein levels , wild-type MEF , Jmjd6 knockout ( KO ) MEF , wild-type JMJD6 overexpression in Jmjd6 KO MEF , inactive mutant JMJD6 overexpression in Jmjd6 KO MEF , wild-type MePCE overexpression in above four MEF cell lines , mutant MePCE ( R169A/R170A/R171A/R172A ) overexpression in wild-type JMJD6 overexpression in Jmjd6 KO MEF , wild-type MePCE with a C-terminal His6 tag overexpression in MePCE KO 293T , and mutant MePCE ( R169A/R170A/R171A/R172A ) with a C-terminal His6 tag overexpression in MePCE KO 293 T cells were grown on 10 cm plates to be harvested and lysed using a standard RIPA buffer mixed with cOmplete Protease inhibitor cocktail ( Roche ) . MEFs were generated from Jmjd6tm1 . 1Gbf knockout mice as previously described ( Böse et al . , 2004; Hahn et al . , 2010 ) . Total cellular extracts in the presence of a protein standard ( Bio-rad ) were resolved by 8–12% gradient SDS-PAGE and transferred to a 0 . 22 μm nitrocellulose membrane and incubated with specific antibodies overnight at 4°C . Antibodies used in this investigation were: Anti-JMJD6 ( Santa Cruz Biotechnology , sc-28348 ) , Anti-LARP7 ( Abcam , ab-134757 ) , Anti-HEXIM1 ( Santa Cruz Biotechnology , sc-390059 ) , Anti-MePCE ( Bethyl Laboratories Inc , A304-184A ) , Anti-Actin ( Santa Cruz Biotechnology , sc-8432 ) , Anti-His ( Santa Cruz Biotechnology , sc-53073 ) , Anti-Pol II CTD ( Gift from Dr . David Bentley ) , Anti-Pol II Ser2p-CTD ( Gift from Dr . David Bentley ) , and Anti-CDK9 ( Santa Cruz Biotechnology sc-13130 ) . Full-length MePCE protein with N-terminal His-tag , mixed with EDTA-free cOmplete protease inhibitor ( Roche ) , αKG , Zn2+ , and HEPES pH 6 . 5 , was titrated with recombinant wild-type JMJD6 and placed in 37°C for 2 hr . The reaction was subject to western blot analysis using monoclonal anti-His antibody ( Santa Cruz Biotechnology , sc-53073 ) . The reaction was reproduced in two separate experiments and the blot bands were quantified using ImageJ . MePCE ( 161-179 ) R171-me2s/C177S peptide , mixed with EDTA-free cOmplete protease inhibitor ( Roche ) , αKG , Zn2+ , and HEPES pH 6 . 5 , was treated with recombinant wild-type JMJD6 , inactive mutant JMJD6 , or peptide alone and placed in 37°C for 2 hr . 1 μL of reaction sample is mixed with 1 μL of a-cyano-4-hydroxycinnamic acid ( 10 mg/ml in 50% ACN , 0 . 1% TFA ) . The mixture is spotted on the MALDI target and allowed to air dry . The sample is analyzed by a Microflex-LRF mass spectrometer ( Bruker Daltonics , Billerica , MA ) in positive ion reflector mode . External calibration is done using a peptide calibration mixture ( 4 to 6 peptides ) on a spot adjacent to the sample . The raw data is processed in the FlexAnalysis software ( version 3 . 4 . 7 , Bruker Daltonics ) and exported in mzXML format . The mzXML files were analyzed on ProteoWizard . Data points were normalized to the intensity of the undigested peptide input and plotted on SigmaPlot v11 . 0 . The reaction was reproduced in three separate experiments . Given the known structure of JMJD6 , where aromatic residues are present in close proximity to the binding pocket ( Y131 , F133 , W174 , W272 ) , the following tryptophan fluorescence assay was deemed suitable for characterizing various peptide binding activity . All the regular and methylated peptides were synthesized by AnaSpec Inc ( Histone 3 ) or Peptide 2 . 0 Inc ( MePCE ) . 5 μM JMJD6 ( 1–343 ) was titrated and equilibrated with fixed concentrations of each peptide respectively , incubated at 25°C for 30 min between each titration intervals , and subject to fluorescence measurement . The buffer used in the fluorescence quenching assay was 100 mM NaCl , 20 mM Tris-HCl pH 6 . 5 , and 0 . 05% Tween-20 . The excitation wavelength of 280 nm and the emission wavelength of 342 nm was used for data collection and recorded with a Fluoromax-3 spectrometer . The titration samples were prepared and analyzed in parallel as duplicates , triplicates , or quadruplicates . All values at different titration points were compiled , normalized against the maximum value obtained prior to titration and averaged . The error bars indicate the normalized minimum and maximum values at any given titration point . The KD for each peptide was calculated by fitting to a four parameter sigmoidal dose-response curve with SigmaPlot v11 . 0 . His-JMJD6 ( 1–343 ) labeled with fluorescent NT-647 dye at a constant concentration of 100 nM was mixed with sixteen serial dilutions ( ~1 . 5 nM–300 μM ) of peptides derived from MePCE ( 154-187 ) , MePCE ( 154-184 ) R170-me2s , MePCE ( 154-184 ) R171-me2s , Histone 3 ( 1-21 ) , Histone 3 ( 1-16 ) R2me2s , C-peptide ( 57-87 ) . C-Peptide ( 57-87 ) was used as a negative control . MST experiment was performed using Monolith NT . 115 ( NanoTemper Technologies ) . His-JMJD6 ( 1–343 ) was titrated with peptides in PBS-T buffer ( 137 mM NaCl , 2 . 7 mM KCl , 10 mM Na2HPO4 , 1 . 8 mM KH2PO4 , and 0 . 05% Tween-20 ) . The change in the fluorescence of bound and unbound labeled His-JMJD6 ( 1–343 ) , ΔF , is indicative of the peptide binding . Plotting ΔF vs . peptide concentration facilitated the generation the dissociation curves , computed by the NTP program . The KD , reflecting the affinity of each of the peptides for His-JMJD6 ( 1–343 ) , was obtained . The error bars indicate the normalized minimum and maximum values at any given titration point . Each experiment was performed in triplicate or quadruplicate . Crystal structure of JMJD6 ( 1–343 ) monomer ( PDB:6MEV ) sans methylarginine was used as the template to build in residues corresponding to MePCE ( 164-178 ) near the catalytic center of JMJD6 in PyMol . No regard for clashes , bonds , or optimization was considered . Structure was exported in . pdb format and uploaded to YASARA energy minimization server ( http://www . yasara . org/minimizationserver . htm ) using default parameters ( Krieger et al . , 2009 ) . Energy-minimized output model was converted to . pdb format and the MePCE ( 164-178 ) residues were minimally adjusted in PyMol to superimpose R171 of MePCE with the methylarginine observed in the crystal structure ( PDB:6MEV ) . Western blot of Pol II Ser2p-CTD and unmodified Pol II CTD in wild-type MEF , Jmjd6 knockout ( KO ) MEF , or wild-type JMJD6 overexpression in Jmjd6 KO MEF was reproduced in four separate experiments . All blot bands were quantified using ImageJ and the band intensity of Pol II Ser2p-CTD was normalized against the band intensity of unmodified Pol II CTD in each respective cell lines . The band intensity of each replicate was normalized against the Pol II Ser2p-CTD level in wild-type MEF , then averaged . The error bars indicate one standard deviation to the observed mean . The P-value was obtained using a paired t-test . RNAs from wild type MEF cells , MEF cells with Jmjd6 knockout , and MEF cells with JMJD6 overexpression cells in Jmjd6 knockout background , respectively , were extracted with Trizol reagent ( ThermoFisher Scientific ) . The extracted RNAs were then sent to Quick biology ( Quick Biology , Pasadena ) for further mRNA purification using oligo-d ( T ) beads . The purified mRNA was then used to build a mRNA library . Mouse Genome mm10 was used as the reference .
More than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II ( Pol II ) . Phosphorylation of Pol II CTD by positive transcription elongation factor b ( P-TEFb ) is a necessary precursor event that enables productive transcription elongation . The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II CTD remains unknown . In this report , we utilize mouse and human models to reveal methylphosphate capping enzyme ( MePCE ) , a core component of the 7SK snRNP complex , as the cognate substrate for Jumonji domain-containing 6 ( JMJD6 ) ’s novel proteolytic function . Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine , enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro , binding assays , and downstream effects of Jmjd6 knockout and overexpression on Pol II CTD phosphorylation . We propose that JMJD6 assists bromodomain containing 4 ( BRD4 ) to recruit P-TEFb to Pol II CTD by disrupting the 7SK snRNP complex .
In animals , an enzyme known as RNA polymerase II ( Pol II for short ) is a key element of the transcription process , whereby the genetic information contained in DNA is turned into messenger RNA molecules in the cells , which can then be translated to proteins . To perform this task , Pol II needs to be activated by a complex of proteins called P-TEFb; however , P-TEFb is usually found in an inactive form held by another group of proteins . Yet , it is unclear how P-TEFb is released and allowed to activate Pol II . Scientists have speculated that another protein called JMJD6 ( Jumonji domain-containing 6 ) is important for P-TEFb to activate Pol II . Various roles for JMJD6 have been proposed , but its exact purpose remains unclear . Recently , two enzymes closely related to JMJD6 were found to be able to make precise cuts in other proteins; Lee , Liu et al . therefore wanted to test whether this is also true of JMJD6 . Experiments using purified JMJD6 showed that it could make a cut in an enzyme called MePCE , which belongs to the group of proteins that hold P-TEFb in its inactive form . Lee , Liu et al . then tested the relationships between these proteins in living human and mouse cells . The levels of activated Pol II were lower in cells without JMJD6 and higher in those without MePCE . Together , the results suggest that JMJD6 cuts MePCE to release P-TEFb , which then activates Pol II . JMJD6 appears to know where to cut by following a specific pattern of elements in the structure of MePCE . When MePCE was mutated so that the pattern changed , JMJD6 was unable to cut it . These results suggest that JMJD6 and related enzymes belong to a new family of proteases , the molecular scissors that can cleave other proteins . The molecules that regulate transcription often are major drug targets , for example in the fight against cancer . Ultimately , understanding the role of JMJD6 might help to identify new avenues for cancer drug development .