Histone acetylation and hSWI/SNF remodeling act in concert to stimulate V(D)J cleavage of nucleosomal DNA

The ordered assembly of immunoglobulin and TCR genes by V(D)J recombination depends on the regulated accessibility of individual loci. We show here that the histone tails and intrinsic nucleosome structure pose significant impediments to V(D)J cleavage. However, alterations to nucleosome structure via histone acetylation or by stable hSWI/SNF-dependent remodeling greatly increase the accessibility of nucleosomal DNA to V(D)J cleavage. Moreover, acetylation and hSWI/SNF remodeling can act in concert on an individual nucleosome to achieve levels of V(D)J cleavage approaching those observed on naked DNA. These results are consistent with a model in which regulated recruitment of chromatin modifying activities is involved in mediating the lineage and stage-specific control of V(D)J recombination.     

Stimulation of V(D)J recombination by histone acetylation

V(D)J recombination assembles functional immunoglobulin and T cell receptor genes from individual gene segments [1]. A common recombination mechanism, initiated by the proteins RAG1 and RAG2 at conserved recombination signal sequences (RSSs), operates at all rearranging loci [2] [3]. It has been proposed that the key regulator of the reaction is 'accessibility' of the RSS within chromatin [4]. Recently, the packaging of RSSs into nucleosomes was shown to inhibit initiation of V(D)J recombination [5] [6]. Nevertheless, the tight tissue specificity of regulation cannot be explained by nucleosome-mediated repression alone because a significant fraction of RSSs would be predicted to lie in linker regions between nucleosomes. Therefore, some aspect of the regulation of the recombination reaction must rely on the disruption of higher-order chromatin structure. Here, we report that histone acetylation directly stimulates the recombination reaction in vivo in the correct cell- and stage-specific manner. Neither expression of RAG genes nor activity of RAG proteins was increased by acetylation. Furthermore, histone acetylation failed to overcome nucleosome-mediated repression of RSS recognition and cleavage in vitro. Our data suggest a role for histone acetylation in stimulating recombination in vivo through disruption of higher-order chromatin structures.     

Changes in histone acetylation are associated with differences in accessibility of V(H) gene segments to V-DJ recombination during B-cell ontogeny and development

Although V(D)J recombination is thought to be regulated by changes in the accessibility of chromatin to the recombinase machinery, the mechanisms responsible for establishing "open" chromatin are poorly understood. We performed a detailed study of the acetylation status of histones associated with 11 V(H) gene segments, their flanking regions, and various intergenic elements during B-cell development and ontogeny, when V(D)J recombination is highly regulated. Histone H4 shows higher and more-regulated acetylation than does histone H3 in the V(H) locus. In adult pro-B cells, V(H) gene segments are acetylated prior to V(D)J rearrangement, with higher acetylation associated with J(H)-distal V(H) gene segments. While large regions of the V(H) locus have similar patterns of histone acetylation, acetylation is narrowly confined to the gene segments, their flanking promoters, and recombinase signal sequence elements. Thus, histone acetylation in the V(H) locus is both locally and globally regulated. Increased histone acetylation accompanies preferential recombination of J(H)-proximal V(H) gene segments in early B-cell ontogeny, and decreased histone acetylation accompanies inhibition of V-DJ recombination in a transgenic model of immunoglobulin heavy-chain allelic exclusion. Thus, changes in histone acetylation appear to be important for both promotion and inhibition of V-DJ rearrangement during B-cell ontogeny and development.     

Histone modifications influence the action of Snf2 family remodelling enzymes by different mechanisms

Alteration of chromatin structure by chromatin modifying and remodelling activities is a key stage in the regulation of many nuclear processes. These activities are frequently interlinked, and many chromatin remodelling enzymes contain motifs that recognise modified histones. Here we adopt a peptide ligation strategy to generate specifically modified chromatin templates and used these to study the interaction of the Chd1, Isw2 and RSC remodelling complexes with differentially acetylated nucleosomes. Specific patterns of histone acetylation are found to alter the rate of chromatin remodelling in different ways. For example, histone H3 lysine 14 acetylation acts to increase recruitment of the RSC complex to nucleosomes. However, histone H4 tetra-acetylation alters the spectrum of remodelled products generated by increasing octamer transfer in trans. In contrast, histone H4 tetra-acetylation was also found to reduce the activity of the Chd1 and Isw2 remodelling enzymes by reducing catalytic turnover without affecting recruitment. These observations illustrate a range of different means by which modifications to histones can influence the action of remodelling enzymes.     

Full-length RAG1 promotes contact with coding and intersignal sequences in RAG protein complexes bound to recombination signals paired in cis

The RAG proteins initiate V(D)J recombination by mediating synapsis and cleavage of two different antigen receptor gene segments through interactions with their flanking recombination signal sequences (RSS). The protein–DNA complexes that support this process have mainly been studied using RAG–RSS complexes assembled using oligonucleotide substrates containing a single RSS that are paired in trans to promote synapsis. How closely these complexes model those formed on longer, more physiologically relevant substrates containing RSSs on the same DNA molecule (in cis) remains unclear. To address this issue, we characterized discrete core and full-length RAG protein complexes bound to RSSs paired in cis. We find these complexes support cleavage activity regulated by V(D)J recombination's ‘12/23 rule’ and exhibit plasticity in RSS usage dependent on partner RSS composition. DNA footprinting studies suggest that the RAG proteins in these complexes mediate more extensive contact with sequences flanking the RSS than previously observed, some of which are enhanced by full-length RAG1, and associated with synapsis and efficient RSS cleavage. Finally, we demonstrate that the RAG1 C-terminus facilitates hairpin formation on long DNA substrates, and full-length RAG1 promotes hairpin retention in the postcleavage RAG complex. These results provide new insights into the mechanism of physiological V(D)J recombination.     

Regulation of V(D)J recombination by nucleosome positioning at recombination signal sequences

A key component in the regulation of V(D)J recombination is control of the accessibility of RAG proteins to recombination signal sequences (RSS). Nucleosomes are known to inhibit this accessibility. We show here that the signal sequence itself represses accessibility by causing nucleosome positioning over the RSS. This positioning is mediated, in vitro and in vivo, by the conserved nonamer of the RSS. Consistent with this strong positioning, nucleosomes at RSSs are resistant to remodelling by nucleosome sliding. In vivo we find that consensus RSSs are preferentially protected, whereas those that lack a consensus nonamer, including some cryptic RSSs, fail to position nucleosomes. Decreased protection of these non-consensus RSSs correlates with their increased use in recombination assays. We therefore suggest that nucleosome positioning by RSSs provides a previously unanticipated level of protection and regulation of V(D)J recombination.     

dMi-2 and ISWI chromatin remodelling factors have distinct nucleosome binding and mobilization properties

Mi-2 and ISWI, two members of the Snf2 superfamily of ATPases, reside in separate ATP-dependent chromatin remodelling complexes. These complexes differ in their biochemical properties and are believed to perform distinct functions in the cell. We have compared the remodelling activity of recombinant Drosophila Mi-2 (dMi-2) with that of recombinant ISWI. Both proteins are nucleosome-stimulated ATPases and promote nucleosome mobilization. However, dMi-2 and ISWI differ in their interaction with nucleosome core particles, in their substrate requirements and in the direction of nucleosome mobilization. We have used antibodies to immobilize a complex containing dMi-2 and the dRPD3 histone deacetylase from Drosophila embryo extracts. This complex shares the nucleosome-stimulated ATPase and nucleosome mobilization properties of recombinant dMi-2, demonstrating that these activities are maintained in a physiological context. Its functional properties distinguish dMi-2 from both SWI2/SNF2 and ISWI, defining a new family of ATP-dependent remodelling machines.     

抗原受体基因重组的表观遗传学调控研究新进展

淋巴细胞是哺乳动物唯一能发生体细胞基因组变化的一类细胞,淋巴细胞在发育过程中通过V(D)J重组获得成熟的特异的抗原受体基因,实现了免疫细胞抗原识别惊人的多样性.关于V(D)J重组的调控机制一直是免疫学研究的重要问题,然而直到将表观遗传学研究引入这一领域,综合遗传学和表观遗传学的研究才真正揭示V(D)J重组精细的调控机制.综述了新近发现的V(D)J重组过程中重要的表观遗传学调控机制,如CpG甲基化,组蛋白修饰,核小体重塑及核拓扑学变化.     

RAG1-mediated ubiquitylation of histone H3 is required for chromosomal V(D)J recombination

RAG1 and RAG2 proteins are key components in V(D)J recombination. The core region of RAG1 is capable of catalyzing the recombination reaction; however, the biological function of non-core RAG1 remains largely unknown. Here, we show that in a murine-model carrying the RAG1 ring-finger conserved cysteine residue mutation (C325Y), V(D)J recombination was abrogated at the cleavage step, and this effect was accompanied by decreased mono-ubiquitylation of histone H3. Further analyses suggest that un-ubiquitylated histone H3 restrains RAG1 to the chromatin by interacting with the N-terminal 218 amino acids of RAG1. Our data provide evidence for a model in which ubiquitylation of histone H3 mediated by the ring-finger domain of RAG1 triggers the release of RAG1, thus allowing its transition into the cleavage phase. Collectively, our findings reveal that the non-core region of RAG1 facilitates chromosomal V(D)J recombination in a ubiquitylation-dependent pathway.     

Nucleosome acetylation sequencing to study the establishment of chromatin acetylation

The establishment of posttranslational chromatin modifications is a major mechanism for regulating how genomic DNA is utilized. However, current in vitro chromatin assays do not monitor histone modifications at individual nucleosomes. Here we describe a strategy, nucleosome acetylation sequencing, that allows us to read the amount of modification at each nucleosome. In this approach, a bead-bound trinucleosome substrate is enzymatically acetylated with radiolabeled acetyl CoA by the SAGA complex from Saccharomyces cerevisae. The product is digested by restriction enzymes that cut at unique sites between the nucleosomes and then counted to quantify the extent of acetylation at each nucleosomal site. We find that we can sensitively, specifically, and reproducibly follow enzyme-mediated nucleosome acetylation. Applying this strategy, when acetylation proceeds extensively, its distribution across nucleosomes is relatively uniform. However, when substrates are used that contain nucleosomes mutated at the major sites of SAGA-mediated acetylation, or that are studied under initial rate conditions, changes in the acetylation distribution can be observed. Nucleosome acetylation sequencing should be applicable to analyzing a wide range of modifications. Additionally, because our trinucleosomes synthesis strategy is highly modular and efficient, it can be used to generate nucleosomal systems in which nucleosome composition differs across the array.     

Determinants of HMGB proteins required to promote RAG1/2-recombination signal sequence complex assembly and catalysis during V(D)J recombination

Efficient assembly of RAG1/2-recombination signal sequence (RSS) DNA complexes that are competent for V(D)J cleavage requires the presence of the nonspecific DNA binding and bending protein HMGB1 or HMGB2. We find that either of the two minimal DNA binding domains of HMGB1 is effective in assembling RAG1/2-RSS complexes on naked DNA and stimulating V(D)J cleavage but that both domains are required for efficient activity when the RSS is incorporated into a nucleosome. The single-domain HMGB protein from Saccharomyces cerevisiae, Nhp6A, efficiently assembles RAG1/2 complexes on naked DNA; however, these complexes are minimally competent for V(D)J cleavage. Nhp6A forms much more stable DNA complexes than HMGB1, and a variety of mutations that destabilize Nhp6A binding to bent microcircular DNA promote increased V(D)J cleavage. One of the two DNA bending wedges on Nhp6A and the analogous phenylalanine wedge at the DNA exit site of HMGB1 domain A were found to be essential for promoting RAG1/2-RSS complex formation. Because the phenylalanine wedge is required for specific recognition of DNA kinks, we propose that HMGB proteins facilitate RAG1/2-RSS interactions by recognizing a distorted DNA structure induced by RAG1/2 binding. The resulting complex must be sufficiently dynamic to enable the series of RAG1/2-mediated chemical reactions on the DNA.     

Recombination centres and the orchestration of V(D)J recombination

The initiation of V(D)J recombination by the recombination activating gene 1 (RAG1) and RAG2 proteins is carefully orchestrated to ensure that antigen receptor gene assembly occurs in the appropriate cell lineage and in the proper developmental order. Here we review recent advances in our understanding of how DNA binding and cleavage by the RAG proteins are regulated by the chromatin structure and architecture of antigen receptor genes. These advances suggest novel mechanisms for both the targeting and the mistargeting of V(D)J recombination, and have implications for how these events contribute to genome instability and lymphoid malignancy.     

Chromatin remodeling at the Ig loci prior to V(D)J recombination.

Rearrangement of Ig H and L chain genes is highly regulated and takes place sequentially during B cell development. Several lines of evidence indicate that chromatin may modulate accessibility of the Ig loci for V(D)J recombination. In this study, we show that remodeling of V and J segment chromatin occurs before V(D)J recombination at the endogenous H and kappa L chain loci. In recombination-activating gene-deficient pro-B cells, there is a reorganization of nucleosomal structure over the H chain J(H) cluster and increased DNase I sensitivity of V(H) and J(H) segments. The pro-B/pre-B cell transition is marked by a decrease in the DNase I sensitivity of V(H) segments and a reciprocal increase in the nuclease sensitivity of Vkappa and Jkappa segments. In contrast, J(H) segments remain DNase I sensitive, and their nucleosomal organization is maintained in mu(+) recombination-activating gene-deficient pre-B cells. These results indicate that initiation of rearrangement is associated with changes in the chromatin structure of both V and J segments, whereas stopping recombination involves changes in only V segment chromatin. We further find an increase in histone H4 acetylation at both the H and kappa L chain loci at the pro-B cell stage. Although histone H4 acetylation appears to be an early change associated with B cell commitment, acetylation alone is not sufficient to promote subsequent modifications in Ig chromatin.     

Effect of CpG methylation on RAG1/RAG2 reactivity: implications of direct and indirect mechanisms for controlling V(D)J cleavage

It has been suggested that DNA methylation/demethylation is involved in regulating V(D)J rearrangement. Although methylated DNA is thought to induce an inaccessible chromatin structure, it is unclear whether DNA methylation can directly control V(D)J recombination independently of chromatin structure. In this study, we tested whether DNA methylation directly affects the reactivity of the RAG1/RAG2 complex. Specific methylation within the heptamer of the recombination signal sequences (RSS) markedly reduced V(D)J cleavage without inhibiting RAG1/RAG2–DNA complex formation. By contrast, methylation at other positions around the RSS did not affect the reactivity of the RAG proteins. The presence of a methyl-CpG binding-domain protein inhibited the binding of the RAG1/RAG2 complex to all the methylated CpG sites that were tested. Our findings suggest that DNA methylation around the RSS may have a previously unexpected function in regulating V(D)J recombination by directly inhibiting V(D)J cleavage, in addition to its general function of inducing an inaccessible chromatin configuration.