ATRX restricts Human Cytomegalovirus (HCMV) viral DNA replication through heterochromatinization and minimizes unpackaged viral genomes.

ATRX limits the accumulation of human cytomegalovirus (HCMV) Immediate Early (IE) proteins at the start of productive, lytic infections, and thus is a part of the cell-intrinsic defenses against infecting viruses. ATRX is a chromatin remodeler and a component of a histone chaperone complex. Therefore, we hypothesized ATRX would inhibit the transcription of HCMV IE genes by increasing viral genome heterochromatinization and decreasing its accessibility. To test this hypothesis, we quantitated viral transcription and genome structure in cells replete with or depleted of ATRX. We found ATRX did indeed limit viral IE transcription, increase viral genome chromatinization, and decrease viral genome accessibility. The inhibitory effects of ATRX extended to Early (E) and Late (L) viral protein accumulation, viral DNA replication, and progeny virion output. However, we found the negative effects of ATRX on HCMV viral DNA replication were independent of its effects on viral IE and E protein accumulation but correlated with viral genome heterochromatinization. Interestingly, the increased number of viral genomes synthesized in ATRX-depleted cells were not efficiently packaged, indicating the ATRX-mediated restriction to HCMV viral DNA replication may benefit productive infection by increasing viral fitness. Our work mechanistically describes the antiviral function of ATRX and introduces a novel, pro-viral role for this protein, perhaps explaining why, unlike during infections with other herpesviruses, it is not directly targeted by a viral countermeasure in HCMV infected cells.

Genomes of the autonomous parvovirus minute virus of mice induce replication stress through RPA exhaustion.

The oncolytic autonomous parvovirus Minute Virus of Mice (MVM) establishes infection in the nuclear environment by usurping host DNA damage signaling proteins in the vicinity of cellular DNA break sites. MVM replication induces a global cellular DNA Damage Response (DDR) that is dependent on signaling by the ATM kinase and inactivates the cellular ATR-kinase pathway. However, the mechanism of how MVM generates cellular DNA breaks remains unknown. Using single molecule DNA Fiber Analysis, we have discovered that MVM infection leads to a shortening of host replication forks as infection progresses, as well as induction of replication stress prior to the initiation of virus replication. Ectopically expressed viral non-structural proteins NS1 and NS2 are sufficient to cause host-cell replication stress, as is the presence of UV-inactivated non-replicative MVM genomes. The host single-stranded DNA binding protein Replication Protein A (RPA) associates with the UV-inactivated MVM genomes, suggesting MVM genomes might serve as a sink for cellular stores of RPA. Overexpressing RPA in host cells prior to UV-MVM infection rescues DNA fiber lengths and increases MVM replication, confirming that MVM genomes deplete RPA stores to cause replication stress. Together, these results indicate that parvovirus genomes induce replication stress through RPA exhaustion, rendering the host genome vulnerable to additional DNA breaks.

The adeno-associated virus 2 genome and Rep 68/78 proteins interact with cellular sites of DNA damage.

Nuclear DNA viruses simultaneously access cellular factors that aid their life cycle while evading inhibitory factors by localizing to distinct nuclear sites. Adeno-associated viruses (AAVs), which are Dependoviruses in the family Parvovirinae, are non-enveloped icosahedral viruses, which have been developed as recombinant AAV vectors to express transgenes. AAV2 expression and replication occur in nuclear viral replication centers (VRCs), which relies on cellular replication machinery as well as coinfection by helper viruses such as adenoviruses or herpesviruses, or exogenous DNA damage to host cells. AAV2 infection induces a complex cellular DNA damage response (DDR), in response to either viral DNA or viral proteins expressed in the host nucleus during infection, where VRCs co-localized with DDR proteins. We have previously developed a modified iteration of a viral chromosome conformation capture (V3C-seq) assay to show that the autonomous parvovirus minute virus of mice localizes to cellular sites of DNA damage to establish and amplify its replication. Similar V3C-seq assays to map AAV2 show that the AAV2 genome co-localized with cellular sites of DNA damage under both non-replicating and replicating conditions. The AAV2 non-structural protein Rep 68/78, also localized to cellular DDR sites during both non-replicating and replicating infections, and also when ectopically expressed. Ectopically expressed Rep could be efficiently re-localized to DDR sites induced by micro-irradiation. Recombinant AAV2 gene therapy vector genomes derived from AAV2 localized to sites of cellular DNA damage to a lesser degree, suggesting that the inverted terminal repeat origins of replication were insufficient for targeting.

The DNA Damage Sensor MRE11 Regulates Efficient Replication of the Autonomous Parvovirus Minute Virus of Mice.

Parvoviruses are single-stranded DNA viruses that utilize host proteins to vigorously replicate in the nuclei of host cells, leading to cell cycle arrest. The autonomous parvovirus, minute virus of mice (MVM), forms viral replication centers in the nucleus which are adjacent to cellular DNA damage response (DDR) sites, many of which are fragile genomic regions prone to undergoing DDR during the S phase. Since the cellular DDR machinery has evolved to transcriptionally suppress the host epigenome to maintain genomic fidelity, the successful expression and replication of MVM genomes at these cellular sites suggest that MVM interacts with DDR machinery distinctly. Here, we show that efficient replication of MVM requires binding of the host DNA repair protein MRE11 in a manner that is independent of the MRE11-RAD50-NBS1 (MRN) complex. MRE11 binds to the replicating MVM genome at the P4 promoter, remaining distinct from RAD50 and NBS1, which associate with cellular DNA break sites to generate DDR signals in the host genome. Ectopic expression of wild-type MRE11 in CRISPR knockout cells rescues virus replication, revealing a dependence on MRE11 for efficient MVM replication. Our findings suggest a new model utilized by autonomous parvoviruses to usurp local DDR proteins that are crucial for viral pathogenesis and distinct from those of dependoparvoviruses, like adeno-associated virus (AAV), which require a coinfected helper virus to inactivate the local host DDR. IMPORTANCE The cellular DNA damage response (DDR) machinery protects the host genome from the deleterious consequences of DNA breaks and recognizes invading viral pathogens. DNA viruses that replicate in the nucleus have evolved distinct strategies to evade or usurp these DDR proteins. We have discovered that the autonomous parvovirus, MVM, which is used to target cancer cells as an oncolytic agent, depends on the initial DDR sensor protein MRE11 to express and replicate efficiently in host cells. Our studies reveal that the host DDR interacts with replicating MVM molecules in ways that are distinct from viral genomes being recognized as simple broken DNA molecules. These findings suggest that autonomous parvoviruses have evolved distinct mechanisms to usurp DDR proteins, which can be used to design potent DDR-dependent oncolytic agents.

HIV-2 inhibits HIV-1 gene expression via two independent mechanisms during cellular co-infection.

IMPORTANCE: Twenty-five years after the first report that HIV-2 infection can reduce HIV-1-associated pathogenesis in dual-infected patients, the mechanisms are still not well understood. We explored these mechanisms in cell culture and showed first that these viruses can co-infect individual cells. Under specific conditions, HIV-2 inhibits HIV-1 through two distinct mechanisms, a broad-spectrum interferon response and an HIV-1-specific inhibition conferred by the HIV-2 TAR. The former could play a prominent role in dually infected individuals, whereas the latter targets HIV-1 promoter activity through competition for HIV-1 Tat binding when the same target cell is dually infected. That mechanism suppresses HIV-1 transcription by stalling RNA polymerase II complexes at the promoter through a minimal inhibitory region within the HIV-2 TAR. This work delineates the sequence of appearance and the modus operandi of each mechanism.

The NS1 protein of the parvovirus MVM Aids in the localization of the viral genome to cellular sites of DNA damage.

The autonomous parvovirus Minute Virus of Mice (MVM) localizes to cellular DNA damage sites to establish and sustain viral replication centers, which can be visualized by focal deposition of the essential MVM non-structural phosphoprotein NS1. How such foci are established remains unknown. Here, we show that NS1 localized to cellular sites of DNA damage independently of its ability to covalently bind the 5' end of the viral genome, or its consensus DNA binding sequence. Many of these sites were identical to those occupied by virus during infection. However, localization of the MVM genome to DNA damage sites occurred only when wild-type NS1, but not its DNA-binding mutant was expressed. Additionally, wild-type NS1, but not its DNA binding mutant, could localize a heterologous DNA molecule containing the NS1 binding sequence to DNA damage sites. These findings suggest that NS1 may function as a bridging molecule, helping the MVM genome localize to cellular DNA damage sites to facilitate ongoing virus replication.

Mutation of a single amino acid of pregnane X receptor switches an antagonist to agonist by altering AF-2 helix positioning.

Pregnane X receptor (PXR) is activated by chemicals to transcriptionally regulate drug disposition and possibly decrease drug efficacy and increase resistance, suggesting therapeutic value for PXR antagonists. We previously reported the antagonist SPA70 and its analog SJB7, which unexpectedly is an agonist. Here, we describe another unexpected observation: mutating a single residue (W299A) within the PXR ligand-binding domain converts SPA70 to an agonist. After characterizing wild-type and W299A PXR activity profiles, we used molecular dynamics simulations to reveal that in wild-type PXR, agonists stabilize the activation function 2 (AF-2) helix in an "inward" position, but SPA70 displaces the AF-2. In W299A, however, SPA70 stabilizes the AF-2 "inward", like agonists. We validated our model by predicting the antagonist SJC2 to be a W299A agonist, which was confirmed experimentally. Our work correlates previously unobserved ligand-induced conformational changes to PXR cellular activity and, for the first time, reveals how PXR antagonists work.

Activation of Mouse Tcrb: Uncoupling RUNX1 Function from Its Cooperative Binding with ETS1.

T lineage commitment requires the coordination of key transcription factors (TFs) in multipotent progenitors that transition them away from other lineages and cement T cell identity. Two important TFs for the multipotent progenitors to T lineage transition are RUNX1 and ETS1, which bind cooperatively to composite sites throughout the genome, especially in regulatory elements for genes involved in T lymphopoiesis. Activation of the TCR ß (Tcrb) locus in committed thymocytes is a critical process for continued development of these cells, and is mediated by an enhancer, Eß, which harbors two RUNX-ETS composite sites. An outstanding issue in understanding T cell gene expression programs is whether RUNX1 and ETS1 have independent functions in enhancer activation that can be dissected from cooperative binding. We now show that RUNX1 is sufficient to activate the endogenous mouse Eß element and its neighboring 25 kb region by independently tethering this TF without coincidental ETS1 binding. Moreover, RUNX1 is sufficient for long-range promoter-Eß looping, nucleosome clearance, and robust transcription throughout the Tcrb recombination center, spanning both DßJß clusters. We also find that a RUNX1 domain, termed the negative regulatory domain for DNA binding, can compensate for the loss of ETS1 binding at adjacent sites. Thus, we have defined independent roles for RUNX1 in the activation of a T cell developmental enhancer, as well as its ability to mediate specific changes in chromatin landscapes that accompany long-range induction of recombination center promoters.

Minute Virus of Mice Inhibits Transcription of the Cyclin B1 Gene during Infection.

Replication of minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus then exploits to prepare the nuclear environment for effective parvovirus takeover. An essential aspect of the MVM-induced DDR is the establishment of a potent premitotic block, which we previously found to be independent of activated p21 and ATR/Chk1 signaling. This arrest, unlike others reported previously, depends upon a significant, specific depletion of cyclin B1 and its encoding RNA, which precludes cyclin B1/CDK1 complex function, thus preventing mitotic entry. We show here that while the stability of cyclin B1 RNA was not affected by MVM infection, the production of nascent cyclin B1 RNA was substantially diminished at late times postinfection. Ectopic expression of NS1 alone did not reduce cyclin B1 expression. MVM infection also reduced the levels of cyclin B1 protein, and RNA levels normally increased in response to DNA-damaging reagents. We demonstrated that at times of reduced cyclin B1 expression during infection, there was a significantly reduced occupancy of RNA polymerase II and the essential mitotic transcription factor FoxM1 on the cyclin B1 gene promoter. Additionally, while total FoxM1 levels remained constant, there was a significant decrease of the phosphorylated, likely active, forms of FoxM1. Targeting of a constitutively active FoxM1 construct or the activation domain of FoxM1 to the cyclin B1 gene promoter via clustered regularly interspaced short palindromic repeats (CRISPR)-enzymatically inactive Cas9 in MVM-infected cells increased both cyclin B1 protein and RNA levels, implicating FoxM1 as a critical target for cyclin B1 inhibition during MVM infection.IMPORTANCE Replication of the parvovirus minute virus of mice (MVM) induces a sustained cellular DNA damage response (DDR) which the virus exploits to prepare the nuclear environment for effective takeover. An essential aspect of the MVM-induced DDR is establishment of a potent premitotic block. This block depends upon a significant, specific depletion of cyclin B1 and its encoding RNA that precludes cyclin B1/CDK1 complex functions necessary for mitotic entry. We show that reduced cyclin B1 expression is controlled primarily at the level of transcription initiation. Additionally, the essential mitotic transcription factor FoxM1 and RNA polymerase II were found to occupy the cyclin B1 gene promoter at reduced levels during infection. Recruiting a constitutively active FoxM1 construct or the activation domain of FoxM1 to the cyclin B1 gene promoter via CRISPR-catalytically inactive Cas9 (dCas9) in MVM-infected cells increased expression of both cyclin B1 protein and RNA, implicating FoxM1 as a critical target mediating MVM-induced cyclin B1 inhibition.

Domain-Specific and Stage-Intrinsic Changes in Tcrb Conformation during Thymocyte Development.

Considerable cross-talk exists between mechanisms controlling genome architecture and gene expression. AgR loci are excellent models for these processes because they are regulated at both conformational and transcriptional levels to facilitate their assembly by V(D)J recombination. Upon commitment to the double-negative stage of T cell development, Tcrb adopts a compact conformation that promotes long-range recombination between Vß gene segments (Trbvs) and their DßJß targets. Formation of a functional VßDßJß join signals for robust proliferation of double-negative thymocytes and their differentiation into double-positive (DP) cells, where Trbv recombination is squelched (allelic exclusion). DP differentiation also is accompanied by decontraction of Tcrb, which has been thought to separate the entire Trbv cluster from DßJß segments (spatial segregation-based model for allelic exclusion). However, DP cells also repress transcription of unrearranged Trbvs, which may contribute to allelic exclusion. We performed a more detailed study of developmental changes in Tcrb topology and found that only the most distal portion of the Trbv cluster separates from DßJß segments in DP thymocytes, leaving most Trbvs spatially available for rearrangement. Preferential dissociation of distal Trbvs is independent of robust proliferation or changes in transcription, chromatin, or architectural factors, which are coordinately regulated across the entire Trbv cluster. Segregation of distal Trbvs also occurs on alleles harboring a functional VßDßJß join, suggesting that this process is independent of rearrangement status and is DP intrinsic. Our finding that most Trbvs remain associated with DßJß targets in DP cells revises allelic exclusion models from their current conformation-dominant to a transcription-dominant formulation.

Unifying model for molecular determinants of the preselection Vß repertoire.

The primary antigen receptor repertoire is sculpted by the process of V(D)J recombination, which must strike a balance between diversification and favoring gene segments with specialized functions. The precise determinants of how often gene segments are chosen to complete variable region coding exons remain elusive. We quantified Vß use in the preselection Tcrb repertoire and report relative contributions of 13 distinct features that may shape their recombination efficiencies, including transcription, chromatin environment, spatial proximity to their DßJß targets, and predicted quality of recombination signal sequences (RSSs). We show that, in contrast to functional Vß gene segments, all pseudo-Vß segments are sequestered in transcriptionally silent chromatin, which effectively suppresses wasteful recombination. Importantly, computational analyses provide a unifying model, revealing a minimum set of five parameters that are predictive of Vß use, dominated by chromatin modifications associated with transcription, but largely independent of precise spatial proximity to DßJß clusters. This learned model-building strategy may be useful in predicting the relative contributions of epigenetic, spatial, and RSS features in shaping preselection V repertoires at other antigen receptor loci. Ultimately, such models may also predict how designed or naturally occurring alterations of these loci perturb the preselection use of variable gene segments.

The Autonomous Parvovirus Minute Virus of Mice Localizes to Cellular Sites of DNA Damage Using ATR Signaling.

Minute Virus of Mice (MVM) is an autonomous parvovirus of the Parvoviridae family that replicates in mouse cells and transformed human cells. MVM genomes localize to cellular sites of DNA damage with the help of their essential non-structural phosphoprotein NS1 to establish viral replication centers. MVM replication induces a cellular DNA damage response that is mediated by signaling through the ATM kinase pathway, while inhibiting induction of the ATR kinase signaling pathway. However, the cellular signals regulating virus localization to cellular DNA damage response sites has remained unknown. Using chemical inhibitors to DNA damage response proteins, we have discovered that NS1 localization to cellular DDR sites is independent of ATM or DNA-PK signaling but is dependent on ATR signaling. Pulsing cells with an ATR inhibitor after S-phase entry leads to attenuated MVM replication. These observations suggest that the initial localization of MVM to cellular DDR sites depends on ATR signaling before it is inactivated by vigorous virus replication.

Activation of HIV-1 proviruses increases downstream chromatin accessibility.

It is unclear how the activation of HIV-1 transcription affects chromatin structure. We interrogated chromatin organization both genome-wide and nearby HIV-1 integration sites using Hi-C and ATAC-seq. In conjunction, we analyzed the transcription of the HIV-1 genome and neighboring genes. We found that long-range chromatin contacts did not differ significantly between uninfected cells and those harboring an integrated HIV-1 genome, whether the HIV-1 genome was actively transcribed or inactive. Instead, the activation of HIV-1 transcription changes chromatin accessibility immediately downstream of the provirus, demonstrating that HIV-1 can alter local cellular chromatin structure. Finally, we examined HIV-1 and neighboring host gene transcripts with long-read sequencing and found populations of chimeric RNAs both virus-to-host and host-to-virus. Thus, multiomics profiling revealed that the activation of HIV-1 transcription led to local changes in chromatin organization and altered the expression of neighboring host genes.

Utilization of Host Cell Chromosome Conformation by Viral Pathogens: Knowing When to Hold and When to Fold.

Though viruses have their own genomes, many depend on the nuclear environment of their hosts for replication and survival. A substantial body of work has therefore been devoted to understanding how viral and eukaryotic genomes interact. Recent advances in chromosome conformation capture technologies have provided unprecedented opportunities to visualize how mammalian genomes are organized and, by extension, how packaging of nuclear DNA impacts cellular processes. Recent studies have indicated that some viruses, upon entry into host cell nuclei, produce factors that alter host chromatin topology, and thus, impact the 3D organization of the host genome. Additionally, a variety of distinct viruses utilize host genome architectural factors to advance various aspects of their life cycles. Indeed, human gammaherpesviruses, known for establishing long-term reservoirs of latent infection in B lymphocytes, utilize 3D principles of genome folding to package their DNA and establish latency in host cells. This manipulation of host epigenetic machinery by latent viral genomes is etiologically linked to the onset of B cell oncogenesis. Small DNA viruses, by contrast, are tethered to distinct cellular sites that support virus expression and replication. Here, we briefly review the recent findings on how viruses and host genomes spatially communicate, and how this impacts virus-induced pathology.

Binding of CCCTC-Binding Factor (CTCF) to the Minute Virus of Mice Genome Is Important for Proper Processing of Viral P4-Generated Pre-mRNAs.

Specific chromatin immunoprecipitation of salt-fractionated infected cell extracts has demonstrated that the CCCTC-binding factor (CTCF), a highly conserved, 11-zinc-finger DNA-binding protein with known roles in cellular and viral genome organization and gene expression, specifically binds the genome of Minute Virus of Mice (MVM). Mutations that diminish binding of CTCF to MVM affect processing of the P4-generated pre-mRNAs. These RNAs are spliced less efficiently to generate the R1 mRNA, and definition of the NS2-specific exon upstream of the small intron is reduced, leading to relatively less R2 and the generation of a novel exon-skipped product. These results suggest a model in which CTCF is required for proper engagement of the spliceosome at the MVM small intron and for the first steps of processing of the P4-generated pre-mRNA.

Viral Chromosome Conformation Capture (V3C) Assays for Identifying Trans-interaction Sites between Lytic Viruses and the Cellular Genome.

The folding mechanisms of the mammalian genome package our genetic material into the nucleus, and in doing so, dictate its appropriate replication and expression. Chromosome conformation capture technology has enabled the dissection of the folding principles of the cellular genome. This has led to a better understanding of the role played by architectural proteins in forming and dissolving 3D-chromatin-structure. These assays are based on the principle of crosslinking distant cellular sites that are proximal to each other in 3D space using formaldehyde followed by digestion of formed hybrid DNA junctions. Invading viruses, such as the lytic parvovirus Minute Virus of Mice (MVM), establish distinct replication centers within the nuclear environment at cellular sites that preferentially undergo DNA damage, but do not integrate into the cellular DNA. We have adapted chromosome conformation capture technology to study the trans-interaction between MVM and the cellular genome, which we have dubbed V3C, which can be extended to a whole-genome analysis we term V3C-seq. This protocol describes the procedure for performing, as well as analyzing V3C-seq assays, and can be adapted for mapping the cellular interaction sites of any non-integrating DNA virus.

Parvovirus minute virus of mice interacts with sites of cellular DNA damage to establish and amplify its lytic infection.

We have developed a generally adaptable, novel high-throughput Viral Chromosome Conformation Capture assay (V3C-seq) for use in trans that allows genome-wide identification of the direct interactions of a lytic virus genome with distinct regions of the cellular chromosome. Upon infection, we found that the parvovirus Minute Virus of Mice (MVM) genome initially associated with sites of cellular DNA damage that in mock-infected cells also exhibited DNA damage as cells progressed through S-phase. As infection proceeded, new DNA damage sites were induced, and virus subsequently also associated with these. Sites of association identified biochemically were confirmed microscopically and MVM could be targeted specifically to artificially induced sites of DNA damage. Thus, MVM established replication at cellular DNA damage sites, which provide replication and expression machinery, and as cellular DNA damage accrued, virus spread additionally to newly damaged sites to amplify infection. MVM-associated sites overlap significantly with previously identified topologically-associated domains (TADs).

Protoparvovirus Interactions with the Cellular DNA Damage Response.

Protoparvoviruses are simple single-stranded DNA viruses that infect many animal species. The protoparvovirus minute virus of mice (MVM) infects murine and transformed human cells provoking a sustained DNA damage response (DDR). This DDR is dependent on signaling by the ATM kinase and leads to a prolonged pre-mitotic cell cycle block that features the inactivation of ATR-kinase mediated signaling, proteasome-targeted degradation of p21, and inhibition of cyclin B1 expression. This review explores how protoparvoviruses, and specifically MVM, co-opt the common mechanisms regulating the DDR and cell cycle progression in order to prepare the host nuclear environment for productive infection.

Regulation of Tcrb Gene Assembly by Genetic, Epigenetic, and Topological Mechanisms.

The adaptive immune system endows mammals with an ability to recognize nearly any foreign invader through antigen receptors that are expressed on the surface of all lymphocytes. This defense network is generated by V(D)J recombination, a set of sequentially controlled DNA cleavage and repair events that assemble antigen receptor genes from physically separated variable (V), joining (J), and sometimes diversity (D) gene segments. The recombination process itself must be stringently regulated to minimize oncogenic translocations involving chromosomes that harbor immunoglobulin and T cell receptor loci. Indeed, V(D)J recombination is controlled at several levels, including tissue-, developmental stage-, allele-, and gene segment-specificity. These levels of control are imposed by a collection of architectural and regulatory elements that are distributed throughout each antigen receptor locus. Together, the genetic elements regulate developmental changes in chromatin, transcription, and locus topology that promote or disfavor long-range recombination. This chapter focuses on the cross talk between these mechanisms at the T cell receptor beta (Tcrb) locus, and how they sculpt a diverse TCRß repertoire while maintaining monospecificity of this antigen receptor on each mature T lymphocyte. We also discuss how insights obtained from studies of Tcrb are more generally relevant to our understanding of gene regulation strategies employed by mammals.

Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element.

Gene regulation relies on dynamic changes in three-dimensional chromatin conformation, which are shaped by composite regulatory and architectural elements. However, mechanisms that govern such conformational switches within chromosomal domains remain unknown. We identify a novel mechanism by which cis-elements promote long-range interactions, inducing conformational changes critical for diversification of the TCRß antigen receptor locus (Tcrb). Association between distal Vß gene segments and the highly expressed DßJß clusters, termed the recombination center (RC), is independent of enhancer function and recruitment of V(D)J recombinase. Instead, we find that tissue-specific folding of Tcrb relies on two distinct architectural elements located upstream of the RC. The first, a CTCF-containing element, directly tethers distal portions of the Vß array to the RC. The second element is a chromatin barrier that protects the tether from hyperactive RC chromatin. When the second element is removed, active RC chromatin spreads upstream, forcing the tether to serve as a new barrier. Acquisition of barrier function by the CTCF element disrupts contacts between distal Vß gene segments and significantly alters Tcrb repertoires. Our findings reveal a separation of function for RC-flanking regions, in which anchors for long-range recombination must be cordoned off from hyperactive RC landscapes by chromatin barriers.