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NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 5 | NOVEMBER 2004 | 869

Post-translational modifications of histones, such asacetylation, phosphorylation and methylation, arethought to be dynamic and reversible. However,whereas the enzymes that help remove acetyl andphosphate groups from histone tails are known,those that counteract methylation have been elusive.But, two separate efforts have now led to the discov-ery that an enzyme, previously known to deiminatehistone Arg residues, also regulates histone Argmethylation.

The enzyme, peptidylarginine deiminase-4(PADI4/PAD4), was known to catalyse the removalof an imino group from peptidyl Arg to producepeptidyl citrulline. In Science, Allis, Coonrod and co-workers now report that, when the substrate ismethylated, PADI4/PAD4 demethyliminates themethylated Arg by releasing a methylamine groupand generating citrulline.

Both groups examined the substrate specificity ofPADI4/PAD4. In Cell, Kouzarides and colleaguesreport that, in vitro, PADI4/PAD4 deiminates Argresidues, specifically Arg2, 8, 17 and 26, in histoneH3. Incidentally, Arg2, 17 and 26 are known sub-strates for the Arg methyltransferase CARM1. TheAllis and Coonrod group showed, both in vitro andin vivo, that PADI4/PAD4 targets include histone-H3Arg8 and 17 and histone-H4 Arg3 (the latter is aknown substrate for a different Arg methyltrans-ferase known as PRMT1).

Using antibodies against specific methylatedArg residues, the Allis and Coonrod team showedthat the PADI4/PAD4 activity does indeed ‘undo’the methylation of Arg substrates. By tracing theradioactivity of the methyl group, they concludedthat the methylimide group might be directlyremoved by demethylimination. Indeed, a dra-matic decrease in the Arg methylation of histoneswas observed in response to PADI4/PAD4 activa-tion in human HL-60 granulocytes. When treatinggranulocytes with PADI4/PAD4 small interfering(si)RNA, the level of histone-H4 Arg3 methylationremained the same and little citrulline wasdetected. This indicated to the Allis and Coonrodgroup that PADI4/PAD4 is the main enzymeresponsible for regulating the levels of histone Argmethylation.

When they treated synthesized peptides withPADI4/PAD4, Kouzarides and co-workers noticedthat dimethylated Arg peptides could not be con-verted to citrulline. Unfortunately, monomethyl Argpeptides were not available, but the decrease inmethylation of histone H3, as measured by an anti-body that is specific for monomethyl Arg, impliesthat only monomethylated Arg residues could betargeted by PADI4/PAD4.

Arg methylation has been linked to transcrip-tional activation in response to hormone induction.Using an oestrogen-responsive promoter fused to areporter gene, the Allis and Coonrod group showedthat the presence of PADI4/PAD4 inhibited the hor-mone-stimulated reporter-gene activity, whereas aPADI4/PAD4 mutant protein failed to do so. As bothgroups confirmed, this transcriptional responsecoincides with the recruitment of PADI4/PAD4 tothe promoter region, an increase in deiminated his-tone levels that is coupled to a decrease in methylatedhistone levels, and with the release of RNA poly-merase II from the promoter.

To probe the mechanism by which PADI4/PAD4antagonizes Arg methylation, the Kouzarides grouptested a tail peptide from histone H3 in which Arg2,Arg8 and Arg17 were replaced by citrulline, and theyfound that the peptide remained unmethylated inthe presence of CARM1. Together with the otherfindings, this indicates two possible mechanisms forPADI4/PAD4 action: first, it might deplete the his-tone-H3 substrate of CARM1, as implied by theKouzarides group; or second, PADI4/PAD4 mightreverse the methylation of monomethylated Arg. Inaddition, this raises the obvious question of howdimethylation of Arg residues is reversed…

Arianne Heinrichs

References and linksORIGINAL RESEARCH PAPERS Wang, Y. et al. Human PAD4regulates histone arginine methylation levels via demethylimination.Science 2 Sept 2004 (doi:10.1126/science.1101400) | Cuthbert, G. L.et al. Histone deimination antagonizes arginine methylation. Cell 118,545–553 (2004)

when expressed in dark-incubatedseedlings, and this transcriptionalactivity was suppressed in the pres-ence of light treatment. PIF1 alsointeracted with the active (Pfr) formof phyA and phyB, the two main phy-tochromes that regulate the ability ofseedlings to undergo ‘greening’ inresponse to light, and the light-induced suppression of PIF1 activityrequired these phy proteins. As PIF1can’t interact with DNA and phyA orphyB concurrently, it seems that thephytoreceptors, when active, mightfunction to modulate the activity ofPIF1 — perhaps by sequestering ordegrading it — so that chlorophyllbiosynthesis can occur in the pres-ence of light. So PIF1 seems to func-tion as “a critical modulator by whichplants optimize chlorophyll biosyn-thesis in response to environmentallight conditions and protect againstaccumulation of potentially toxiclevels of intermediates.”

Katrin BussellReferences and links

ORIGINAL RESEARCH PAPER Huq, E. et al.PHYTOCHROME-INTERACTING FACTOR 1 is acritical bHLH regulator of chlorophyll biosynthesis.Science 305, 1937–1941 (2004) FURTHER READING Quail, P. H. Phytochromephotosensory signalling networks. Nature Rev.Mol. Cell Biol. 3, 85–93 (2002)

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