Search

ATRX is a chromatin remodeler, which is mutated in ATRX syndrome, a neurodevelopmental disorder. ATRX mutations that alter histone binding or chromatin remodeling activities cluster in the PHD finger or the helicase domain respectively. Using engineered mouse embryonic stem cells that exclusively express ATRX protein with mutations in the PHD finger (PHDmut) or helicase domains (K1584R), we examine how specific ATRX mutations affect neurodifferentiation. ATRX PHDmut and K1584R proteins interact with the DAXX histone chaperone but show reduced localization to pericentromeres. Neurodifferentiation is both delayed and compromised in PHDmut and K1584R, and manifest differently from complete ATRX loss. We observe reduced enrichment of PHDmut protein to ATRX targets, while K1584R accumulates at these sites. Interestingly, ATRX mutations have distinct effects on the genome-wide localization of the polycomb repressive complex 2 (PRC2), with PHDmut and ATRX knockout showing reduced PRC2 binding at polycomb targets and K1584R showing loss at some sites and gains at others. Notably, each mutation associated with unique gene signatures, suggesting distinct pathways leading to impaired neurodifferentiation. Our results indicate that the histone binding and chromatin remodeling functions of ATRX play non-redundant roles in neurodevelopment, and when mutated lead to ATRX syndrome through separate regulatory pathways., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A large fraction of the mammalian genome is transcribed into long noncoding RNAs. The RNAs remain largely uncharacterized as the field awaits new technologies to aid functional analysis. Here, we describe a unique use of locked nucleic acids (LNAs) for studying nuclear long noncoding RNA, an RNA subclass that has been less amenable to traditional knockdown techniques. We target LNAs at Xist RNA and show displacement from the X chromosome with fast kinetics. Xist transcript stability is not affected. By targeting different Xist regions, we identify a localization domain and show that polycomb repressive complex 2 (PRC2) is displaced together with Xist. Thus, PRC2 depends on RNA for both initial targeting to and stable association with chromatin. H3K27-trimethyl marks and gene silencing remain stable. Time-course analysis of RNA relocalization suggests that Xist and PRC2 bind to different regions of the X at the same time but do not reach saturating levels immediately. Thus, LNAs provide a tool for studying an emerging class of regulatory RNA and offer a window of opportunity to target epigenetic modifications with possible therapeutic applications.

The histone methyl transferase PR-Set7 mediates histone H4 Lys 20 methylation, a mark of constitutive and facultative heterochromatin. We isolated a null mutation in Drosophila PR-Set7 that suppresses position effect variegation, indicating that PR-Set7 indeed functions in silencing general gene expression. In PR-Set7 larval leg and eye discs, the number of cells is lower than normal, and the DNA content in these cells is significantly increased. These data show that PR-Set7-dependent methylation is essential for the process of mitosis. The methylation mark is highly stable and is maintained even in the absence of PR-Set7 protein.

A fascinating aspect of how chromatin structure impacts on gene expression and cellular identity is the transmission of information from mother to daughter cells, independently of the primary DNA sequence. This epigenetic information seems to be contained within the covalent modifications of histone polypeptides and the distinctive characteristics of variant histone subspecies. There are specific deposition pathways for some histone variants, which provide invaluable mechanistic insights into processes whereby the major histones are exchanged for their more specialized counterparts.

Histone H3 tail modifications are among the earliest chromatin changes in the X-chromosome inactivation process. In this study we investigated the relative profiles of two important repressive marks on the X chromosome: methylation of H3 lysine 9 (K9) and 27 (K27). We found that both H3K9 dimethylation and K27 trimethylation characterize the inactive X in somatic cells and that their relative kinetics of enrichment on the X chromosome as it undergoes inactivation are similar. However, dynamic changes of H3K9 and H3K27 methylation on the inactivating X chromosome compared to the rest of the genome are distinct, suggesting that these two modifications play complementary and perhaps nonredundant roles in the establishment and/or maintenance of X inactivation. Furthermore, we show that a hotspot of H3K9 dimethylation 5' to Xist also displays high levels of H3 tri-meK27. However, analysis of this region in G9a mutant embryonic stem cells shows that these two methyl marks are dependent on different histone methyltransferases.

Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. Here we show that H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, we identified Suv4-20h1 and Suv4-20h2 as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, the Suv4-20 homolog is a novel PEV modifier to regulate position-effect variegation. Together, our data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin.

We describe distinct patterns of histone methylation during human cell cycle progression. Histone H4 methyltransferase activity was found to be cell cycle-regulated, consistent with increased H4 Lys 20 methylation at mitosis. This increase closely followed the cell cycle-regulated expression of the H4 Lys 20 methyltransferase, PR-Set7. Localization of PR-Set7 to mitotic chromosomes and subsequent increase in H4 Lys 20 methylation were inversely correlated to transient H4 Lys 16 acetylation in early S-phase. These data suggest that H4 Lys 20 methylation by PR-Set7 during mitosis acts to antagonize H4 Lys 16 acetylation and to establish a mechanism by which this mark is epigenetically transmitted.

We have purified a human histone H4 lysine 20 methyltransferase and cloned the encoding gene, PR/SET07. A mutation in Drosophila pr-set7 is lethal: second instar larval death coincides with the loss of H4 lysine 20 methylation, indicating a fundamental role for PR-Set7 in development. Transcriptionally competent regions lack H4 lysine 20 methylation, but the modification coincided with condensed chromosomal regions on polytene chromosomes, including chromocenter and euchromatic arms. The Drosophila male X chromosome, which is hyperacetylated at H4 lysine 16, has significantly decreased levels of lysine 20 methylation compared to that of females. In vitro, methylation of lysine 20 and acetylation of lysine 16 on the H4 tail are competitive. Taken together, these results support the hypothesis that methylation of H4 lysine 20 maintains silent chromatin, in part, by precluding neighboring acetylation on the H4 tail.

A novel histone methyltransferase, termed Set9, was isolated from human cells. Set9 contains a SET domain, but lacks the pre- and post-SET domains. Set9 methylates specifically lysine 4 (K4) of histone H3 (H3-K4) and potentiates transcription activation. The histone H3 tail interacts specifically with the histone deacetylase NuRD complex. Methylation of histone H3-K4 by Set9 precludes the association of NuRD with the H3 tail. Moreover, methylation of H3-K4 impairs Suv39h1-mediated methylation at K9 of H3 (H3-K9). The interplay between the Set9 and Suv39h1 histone methyltransferases is specific, as the methylation of H3-K9 by the histone methyltransferase G9a was not affected by Set9 methylation of H3-K4. Our studies suggest that Set9-mediated methylation of H3-K4 functions in transcription activation by competing with histone deacetylases and by precluding H3-K9 methylation by Suv39h1. Our results suggest that the methylation of histone tails can have distinct effects on transcription, depending on its chromosomal location, the combination of posttranslational modifications, and the enzyme (or protein complex) involved in the particular modification.