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research highlights

EPIGENETICS Writing the code

Recruiting modifiers to a spe- modifier protein 1 (HP1) HP1 cific locus allows the regulation of het- fused to a recruiter protein. Adding a small erochromatin formation in living cells. molecule facilitates the rapid association of Genetic memory does not live by basic anchor with recruiter and brings HP1 to Oct4 Drug DNA sequence alone. Decades of research the targeted locus on the DNA. promoter have established the importance of his- HP1 binds the 9 on histone 3 and tones and their various post-translational recruits histone methylases, which add

modifications in determining gene activa- methyl groups to adjacent H3 molecules, Vicari Katie tion and passing these changes on to the thus spreading the mark on next generation of cells. chromatin. The chromatin in vivo assay allows recruitment Some of these modifications, such as After the scientists had ascertained of chromatin modifiers to a specific locus in live the triple on lysine 9 of his- that their modified Oct4 allele itself had cells. tone 3, H3K9me3 for short, have well- no effect on the behavior of the ESCs— established roles in gene silencing and because Oct4 is haplosufficient, ESCs can heterochromatin formation. A lot is dispense with one allele—they induced Crabtree sees more potential applications known about the enzymes that methylate HP1 recruitment and monitored silenc- of the chromatin in vivo assay. “The tech- the histone, but the timing and regulation ing of GFP expression and the spreading of nique allows the study of the mechanis- of this methylation process in the context H3K9me3 via chromatin immunoprecipi- tic underpinnings of reprogramming,” of a living cell remain elusive. tation. Courtney Hodges, a postdoctoral he notes. Gerald Crabtree from Stanford fellow working with Crabtree, derived a For Crabtree, one of the big lessons of University was frustrated with trying to mathematical model that allowed them to this work lay not so much in what they dis- use in vitro assays calculate the rate of H3K9me3 spreading. covered but in what they did not see. “It

© 2012 Nature America, Inc. All rights reserved. America, Inc. © 2012 Nature to address these questions. “You are more After 3 days of permanent HP1 recruit- allows us to predict where the holes are,” or less stuck with artificial templates: ment, they saw a population of cells with he says. When they first developed the short pieces of DNA that bear no relation- a completely silenced Oct4 promoter. In model for H3K9me3 spreading, Crabtree

npg ship to the actual template, which has dif- addition to H3K9me3, the scientists also recalls being pleased with the result; how- ferent histone modifications in different noted an increase in DNA methylation. ever, it soon became clear that during the tissues,” he says. “I was surprised,” recalls Crabtree, “that first few hours of HP1 recruitment, some- Instead he wanted an approach that recruiting HP1 also recruits the [DNA] thing was going on that they did not under- would allow him to quickly add and methylation system, but there is evidence stand. HP1 is recruited rapidly, but the first remove a chromatin modification from a that the cross-talk exists and reinforces H3K9 methylation mark is much slower locus of choice in a living cell. Together the degree of repression. It makes it more to appear. Something seems to occupy with postdoctoral fellows Oliver Bell and s t a b l e .” this locus that first needs to be removed. Nathaniel Hathaway, Crabtree brought this Oct4 expression is silenced in differenti- Crabtree calls this factor the ‘nonvisible idea to life in the form of their chromatin ated cells, but Crabtree and his team wanted part’ of their assay and has some ideas, in vivo assay. to know whether the heterochromatin mark such as using RNA interference or small Working in mouse embryonic stem cells could be erased by active . molecule screens, for figuring out what (ESCs), they targeted the promoter of one They generated a mouse from their modi- is happening during the first few hours of Oct4 allele—one of four genes essential for fied ESCs, isolated embryonic fibroblasts chromatin silencing. renewal—with landing sites for and targeted a transcriptional activator to Now that the can be active- either a zinc-finger protein or the DNA the silenced Oct4 locus. They saw reactiva- ly (re)written, much waits to be explored. binding protein Gal4, and they replaced the tion of GFP expression after only 24 hours; Nicole Rusk first exon of Oct4 with GFP to report gene after 5 days, 10% of the cells were GFP RESEARCH PAPERS expression. Next they expressed two fusion positive. Such reactivation of silent loci is Hathaway, N.A. et al. Dynamics and memory of proteins: a zinc-finger protein combined essential during reprogramming when dif- heterochromatin in living cells. Cell 149, 1447–1460 with an anchor protein and the histone ferentiated cells regain a pluripotent state. (2012).

nature methods | VOL.9 NO.8 | AUGUST 2012 | 777