Nucleosomal asymmetry: a novel mechanism to regulate nucleosome function

Nucleosomes constitute the fundamental building blocks of chromatin. They are comprised of DNA wrapped around a histone octamer formed of two copies each of the four core histones H2A, H2B, H3, and H4. Nucleosomal histones undergo a plethora of posttranslational modifications that regulate gene expression and other chromatin-templated processes by altering chromatin structure or by recruiting effector proteins. Given their symmetric arrangement, the sister histones within a nucleosome have commonly been considered to be equivalent and to carry the same modifications. However, it is now clear that nucleosomes can exhibit asymmetry, combining differentially modified sister histones or different variants of the same histone within a single nucleosome. Enabled by the development of novel tools that allow generating asymmetrically modified nucleosomes, recent biochemical and cell-based studies have begun to shed light on the origins and functional consequences of nucleosomal asymmetry. These studies indicate that nucleosomal asymmetry represents a novel regulatory mechanism in the establishment and functional readout of chromatin states. Asymmetry expands the combinatorial space available for setting up complex sets of histone marks at individual nucleosomes, regulating multivalent interactions with histone modifiers and readers. The resulting functional consequences of asymmetry regulate transcription, poising of developmental gene expression by bivalent chromatin, and the mechanisms by which oncohistones deregulate chromatin states in cancer. Here, we review recent progress and current challenges in uncovering the mechanisms and biological functions of nucleosomal asymmetry

Two-factor authentication underpins the precision of the piRNA pathway

The PIWI-interacting RNA (piRNA) pathway guides the DNA methylation of young, active transposons during germline development in male mice1. piRNAs tether the PIWI protein MIWI2 (PIWIL4) to the nascent transposon transcript, resulting in DNA methylation through SPOCD1 (refs. 2–5). Transposon methylation requires great precision: every copy needs to be methylated but off-target methylation must be avoided. However, the underlying mechanisms that ensure this precision remain unknown. Here, we show that SPOCD1 interacts directly with SPIN1 (SPINDLIN1), a chromatin reader that primarily binds to H3K4me3-K9me3 (ref. 6). The prevailing assumption is that all the molecular events required for piRNA-directed DNA methylation occur after the engagement of MIWI2. We find that SPIN1 expression precedes that of both SPOCD1 and MIWI2. Furthermore, we demonstrate that young LINE1 copies, but not old ones, are marked by H3K4me3, H3K9me3 and SPIN1 before the initiation of piRNA-directed DNA methylation. We generated a Spocd1 separation-of-function allele in the mouse that encodes a SPOCD1 variant that no longer interacts with SPIN1. We found that the interaction between SPOCD1 and SPIN1 is essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 elements. We propose that piRNA-directed LINE1 DNA methylation requires a developmentally timed two-factor authentication process. The first authentication is the recruitment of SPIN1–SPOCD1 to the young LINE1 promoter, and the second is MIWI2 engagement with the nascent transcript. In summary, independent authentication events underpin the precision of piRNA-directed LINE1 DNA methylation

A coherent feed-forward loop drives vascular regeneration in damaged aerial organs of plants growing in a normal developmental context

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.Peer reviewe

The histone H3.1 variant regulates TONSOKU-mediated DNA repair during replication

The tail of replication-dependent histone H3.1 varies from that of replication-independent H3.3 at the amino acid located at position 31 in plants and animals, but no function has been assigned to this residue to demonstrate a unique and conserved role for H3.1 during replication. Here, we show that TONSOKU (TSK/TONSL), which rescues broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat domain. Our results indicate that genomic instability in the absence of ATXR5/ATXR6-catalyzed H3K27me1 in plants depends on H3.1, TSK and DNA polymerase theta (Pol θ). Overall, this work reveals an H3.1-specific function during replication and the common strategy used in multicellular eukaryotes for regulating post-replicative chromatin maturation and TSK, which relies on histone mono-methyltransferases and reading the H3.1 variant

Roles of nucleosome asymmetry and KAT6B-mediated histone acetylation in the regulation of bivalent promoters

Histone modifications are key regulators of gene expression. Nucleosomes can be modified both symmetrically and asymmetrically, carrying a specific modification on one or both sister histones within a nucleosome. Bivalent nucleosomes are a special case of asymmetric nucleosomes, carrying the active mark histone H3 lysine 4 trimethylation (H3K4me3) and the repressive mark H3K27me3 in an asymmetric fashion. Bivalency is postulated to fine-tune gene expression during development, however, the mechanisms by which bivalent genes are poised for expression are still not well understood. To determine how nucleosomal asymmetry regulates histone mark readout, I generated asymmetric nucleosomes and performed nucleosome pulldown assays. H3K4me3 binders exhibited differential binding to symmetric and asymmetric nucleosomes, whereas more abundant H3K27me3 binders such as Polycomb complex subunits were recruited similarly to both symmetric and asymmetric nucleosomes. While nucleosomal asymmetry affects reader domains for H3K4me3 and H3K27me3 similarly, differential reader abundance and basal nucleosome affinity of repressive binders drive differential enrichment of active and repressive binders on asymmetric nucleosomes in embryonic stem cells. Accordingly, asymmetric bivalent nucleosomes attract H3K27me3 binders but fail to enrich activating H3K4me3 binders. These findings show that nucleosomal asymmetry is critical for histone mark readout and function, providing a mechanism for differential recruitment of active and repressive complexes at bivalent domains. Bivalent nucleosomes also attract specific binders that are not enriched by the individual marks, including the histone acetyltransferase complex KAT6B. This finding suggests that KAT6B recruitment involves a combinatorial readout of H3K4me3, unmodified H3K4 (H3K4un), and H3K27me3. However, KAT6B does not possess any known binding domains for H3K27me3. I tested whether the catalytic MYST domain interacts with H3K27me3 in the context of acetylating H3K23 or whether KAT6B interacts with PRC1. MYST domain indeed exhibited a modestly increased affinity for nucleosomes in the presence of H3K27me3. In addition, proximity labelling suggests that KAT6B interacts with PRC1, providing an additional mechanism for interaction recruitment of KAT6B to bivalent nucleosomes. This interaction was further confirmed via immunoprecipitation. These observations suggests that a combination of direct KAT6B interaction with H3K27me3 and indirect interaction via PRC1 mediates its enhanced recruitment at bivalent genes. The presence of KAT6B at bivalent genes suggests that its marks H3K23ac and H3K14ac regulate readout of bivalent nucleosomes. Nucleosome pulldown-based proteomics analysis revealed that H3K23ac enriches the chromatin remodellers SWI/SNF and ISWI at bivalent nucleosomes, suggesting that H3K23ac promotes resolution of bivalency towards activation during differentiation via recruitment of chromatin remodellers. While further investigation is required to understand the underlying mechanisms, these studies explore how nucleosome asymmetry, histone acetylation and chromatin remodellers work in concert to shape the regulatory landscape at bivalent domains

The histone H3.1 variant regulates TONSOKU-mediated DNA repair during replication

The tail of replication-dependent histone H3.1 varies from that of replication-independent H3.3 at the amino acid located at position 31 in plants and animals, but no function has been assigned to this residue to demonstrate a unique and conserved role for H3.1 during replication. We found that TONSOKU (TSK/TONSL), which rescues broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat domain. Our results indicate that genomic instability in the absence of ATXR5/ATXR6-catalyzed histone H3 lysine 27 monomethylation in plants depends on H3.1, TSK, and DNA polymerase theta (Pol θ). This work reveals an H3.1-specific function during replication and a common strategy used in multicellular eukaryotes for regulating post-replicative chromatin maturation and TSK, which relies on histone monomethyltransferases and reading of the H3.1 variant.</jats:p

A coherent feed forward loop drives vascular regeneration in damaged aerial organs growing in normal developmental-context

Aerial organs of plants being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaf and stem to study the reunion between mechanically disconnected tissues. We show that PLETHORA(PLT)/ AINTEGUMENTA(ANT) genes, which encodes stem cell promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. Both PLT and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT mediated regeneration response, leaf ground tissue cells can neither acquire early vascular identity marker ATHB8, nor properly polarize auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaf. We reveal the mechanisms of vascular regeneration in plants and distinguishes the wound repair ability of the tissue from its formation during normal development.</jats:p