RNA Helicase DDX1 Converts RNA G-Quadruplex Structures into R-Loops to Promote IgH Class Switch Recombination.

Class switch recombination (CSR) at the immunoglobulin heavy-chain (IgH) locus is associated with the formation of R-loop structures over switch (S) regions. While these often occur co-transcriptionally between nascent RNA and template DNA, we now show that they also form as part of a post-transcriptional mechanism targeting AID to IgH S-regions. This depends on the RNA helicase DDX1 that is also required for CSR in vivo. DDX1 binds to G-quadruplex (G4) structures present in intronic switch transcripts and converts them into S-region R-loops. This in turn targets the cytidine deaminase enzyme AID to S-regions so promoting CSR. Notably R-loop levels over S-regions are diminished by chemical stabilization of G4 RNA or by the expression of a DDX1 ATPase-deficient mutant that acts as a dominant-negative protein to reduce CSR efficiency. In effect, we provide evidence for how S-region transcripts interconvert between G4 and R-loop structures to promote CSR in the IgH locus.

Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senescence.

Extensive tracts of the mammalian genome that lack protein-coding function are still transcribed into long noncoding RNA. While these lncRNAs are generally short lived, length restricted, and non-polyadenylated, how their expression is distinguished from protein-coding genes remains enigmatic. Surprisingly, depletion of the ubiquitous Pol-II-associated transcription elongation factor SPT6 promotes a redistribution of H3K36me3 histone marks from active protein coding to lncRNA genes, which correlates with increased lncRNA transcription. SPT6 knockdown also impairs the recruitment of the Integrator complex to chromatin, which results in a transcriptional termination defect for lncRNA genes. This leads to the formation of extended, polyadenylated lncRNAs that are both chromatin restricted and form increased levels of RNA:DNA hybrid (R-loops) that are associated with DNA damage. Additionally, these deregulated lncRNAs overlap with DNA replication origins leading to localized DNA replication stress and a cellular senescence phenotype. Overall, our results underline the importance of restricting lncRNA expression.

The DNA-binding protein CTCF limits proximal Vκ recombination and restricts κ enhancer interactions to the immunoglobulin κ light chain locus.

Regulation of immunoglobulin (Ig) V(D)J gene rearrangement is dependent on higher-order chromatin organization. Here, we studied the in vivo function of the DNA-binding zinc-finger protein CTCF, which regulates interactions between enhancers and promoters. By conditional deletion of the Ctcf gene in the B cell lineage, we demonstrate that loss of CTCF allowed Ig heavy chain recombination, but pre-B cell proliferation and differentiation was severely impaired. In the absence of CTCF, the Igκ light chain locus showed increased proximal and reduced distal Vκ usage. This was associated with enhanced proximal Vκ and reduced Jκ germline transcription. Chromosome conformation capture experiments demonstrated that CTCF limits interactions of the Igκ enhancers with the proximal V(κ) gene region and prevents inappropriate interactions between these strong enhancers and elements outside the Igκ locus. Thus, although Ig gene recombination can occur in the absence of CTCF, it is a critical factor determining Vκ segment choice for recombination.

Pre-B cell receptor signaling induces immunoglobulin κ locus accessibility by functional redistribution of enhancer-mediated chromatin interactions.

During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline V(κ) transcription. To investigate whether pre-BCR signaling modulates V(κ) accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb V(κ) region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3'κ enhancer with V(κ) genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and V(κ) genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used V(κ) genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the V(κ) region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the V(κ) region, whereby the two enhancers play distinct roles.

Allelic exclusion of the immunoglobulin heavy chain locus is independent of its nuclear localization in mature B cells.

In developing B cells, the immunoglobulin heavy chain (IgH) locus is thought to move from repressive to permissive chromatin compartments to facilitate its scheduled rearrangement. In mature B cells, maintenance of allelic exclusion has been proposed to involve recruitment of the non-productive IgH allele to pericentromeric heterochromatin. Here, we used an allele-specific chromosome conformation capture combined with sequencing (4C-seq) approach to unambigously follow the individual IgH alleles in mature B lymphocytes. Despite their physical and functional difference, productive and non-productive IgH alleles in B cells and unrearranged IgH alleles in T cells share many chromosomal contacts and largely reside in active chromatin. In brain, however, the locus resides in a different repressive environment. We conclude that IgH adopts a lymphoid-specific nuclear location that is, however, unrelated to maintenance of allelic exclusion. We additionally find that in mature B cells-but not in T cells-the distal VH regions of both IgH alleles position themselves away from active chromatin. This, we speculate, may help to restrict enhancer activity to the productively rearranged VH promoter element.

DNA-binding factor CTCF and long-range gene interactions in V(D)J recombination and oncogene activation.

Regulation of V(D)J recombination events at immunoglobulin (Ig) and T-cell receptor loci in lymphoid cells is complex and achieved via changes in substrate accessibility. Various studies over the last year have identified the DNA-binding zinc-finger protein CCCTC-binding factor (CTCF) as a crucial regulator of long-range chromatin interactions. CTCF often controls specific interactions by preventing inappropriate communication between neighboring regulatory elements or independent chromatin domains. Although recent gene targeting experiments demonstrated that the presence of the CTCF protein is not required for the process of V(D)J recombination per se, CTCF turned out to be essential to control order, lineage specificity and to balance the Ig V gene repertoire. Moreover, CTCF was shown to restrict activity of κ enhancer elements to the Ig κ locus. In this review, we discuss CTCF function in the regulation of V(D)J recombination on the basis of established knowledge on CTCF-mediated chromatin loop domains in various other loci, including the imprinted H19-Igf2 locus as well as the complex ß-globin, MHC class II and IFN-γ loci. Moreover, we discuss that loss of CTCF-mediated restriction of enhancer activity may well contribute to oncogenic activation, when in chromosomal translocations Ig enhancer elements and oncogenes appear in a novel genomic context.

CTCF regulates cell cycle progression of alphabeta T cells in the thymus.

The 11-zinc finger protein CCCTC-binding factor (CTCF) is a highly conserved protein, involved in imprinting, long-range chromatin interactions and transcription. To investigate its function in vivo, we generated mice with a conditional Ctcf knockout allele. Consistent with a previous report, we find that ubiquitous ablation of the Ctcf gene results in early embryonic lethality. Tissue-specific inactivation of CTCF in thymocytes specifically hampers the differentiation of alphabeta T cells and causes accumulation of late double-negative and immature single-positive cells in the thymus of mice. These cells are normally large and actively cycling, and contain elevated amounts of CTCF. In Ctcf knockout animals, however, these cells are small and blocked in the cell cycle due to increased expression of the cyclin-CDK inhibitors p21 and p27. Taken together, our results show that CTCF is required in a dose-dependent manner and is involved in cell cycle progression of alphabeta T cells in the thymus. We propose that CTCF positively regulates cell growth in rapidly dividing thymocytes so that appropriate number of cells are generated before positive and negative selection in the thymus.

Critical role for the transcription regulator CCCTC-binding factor in the control of Th2 cytokine expression.

Differentiation of naive CD4+ cells into Th2 cells is accompanied by chromatin remodeling at the Th2 cytokine locus allowing the expression of the IL-4, IL-5, and IL-13 genes. In this report, we investigated the role in Th2 differentiation of the transcription regulator CCCTC-binding factor (CTCF). Chromatin immunoprecipitation analysis revealed multiple CTCF binding sites in the Th2 cytokine locus. Conditional deletion of the Ctcf gene in double-positive thymocytes allowed development of peripheral T cells, but their activation and proliferation upon anti-CD3/anti-CD28 stimulation in vitro was severely impaired. Nevertheless, when TCR signaling was circumvented with phorbol ester and ionomycin, we observed proliferation of CTCF-deficient T cells, enabling the analysis of Th2 differentiation in vitro. We found that in CTCF-deficient Th2 polarization cultures, transcription of IL-4, IL-5, and IL-13 was strongly reduced. By contrast, CTCF deficiency had a moderate effect on IFN-gamma production in Th1 cultures and IL-17 production in Th17 cultures was unaffected. Consistent with a Th2 cytokine defect, CTCF-deficient mice had very low levels of IgG1 and IgE in their serum, but IgG2c was close to normal. In CTCF-deficient Th2 cultures, cells were polarized toward the Th2 lineage, as substantiated by induction of the key transcriptional regulators GATA3 and special AT-rich binding protein 1 (SATB1) and down-regulation of T-bet. Also, STAT4 expression was low, indicating that in the absence of CTCF, GATA3 still operated as a negative regulator of STAT4. Taken together, these findings show that CTCF is essential for GATA3- and SATB1-dependent regulation of Th2 cytokine gene expression.

Biosynthesis of histone messenger RNA employs a specific 3' end endonuclease.

Replication-dependent (RD) core histone mRNA produced during S-phase is the only known metazoan protein-coding mRNA presenting a 3' stem-loop instead of the otherwise universal polyA tail. A metallo ß-lactamase (MBL) fold enzyme, cleavage and polyadenylation specificity factor 73 (CPSF73), is proposed to be the sole endonuclease responsible for 3' end processing of both mRNA classes. We report cellular, genetic, biochemical, substrate selectivity, and crystallographic studies providing evidence that an additional endoribonuclease, MBL domain containing protein 1 (MBLAC1), is selective for 3' processing of RD histone pre-mRNA during the S-phase of the cell cycle. Depletion of MBLAC1 in cells significantly affects cell cycle progression thus identifying MBLAC1 as a new type of S-phase-specific cancer target.

Dynamic Control of Long-Range Genomic Interactions at the Immunoglobulin κ Light-Chain Locus.

The Igκ locus, which is spread over 3Mb of genomic DNA and contains >100 variable (V) genes, serves as an important model system to study long-range chromatin interactions. Here, we will discuss how in developing B cells in the bone marrow the accessibility of individual Vκ segments is controlled by many lineage-specific and ubiquitously expressed transcription factors that act on various cis-regulatory elements, including promoters, enhancers, and insulators. This dynamic control furthermore involves changes in subnuclear localization, histone modification, DNA demethylation, and three-dimensional locus compaction. In pro-B cells, the Igκ locus adopts a poised conformation as full contraction has been achieved and many key transcription factors already occupy the locus. Subsequently, the combined activation of pre-B cell antigen receptor signaling pathways and attenuation of IL-7R signaling in small resting pre-B cells dramatically modifies the transcription factor landscape, supporting the induction of monoallelic Igκ gene rearrangements. Hereby, the intronic and 3' Igκ enhancer elements coordinately focus their activities in the Vκ region toward frequently used Vκ genes. Recent work has drawn attention to the intriguing role of the CTCF-associated regulatory elements Cer and Sis, which are located in the Vκ-Jκ intervening region and control Igκ locus contraction and Vκ repertoire diversity. This involves CTCF-mediated locus insulation, restricting enhancer activity to the Vκ region and suppressing the preferential recombination to proximal Vκ genes. A picture emerges in which the dynamic control of long-range genomic interactions ensures correct timing of Igκ locus recombination and provides appropriate opportunities for individual Vκ gene segments to engage in Vκ-Jκ rearrangement.

The DNA-binding factor Ctcf critically controls gene expression in macrophages.

Macrophages play an important role in immunity and homeostasis. Upon pathogen recognition via specific receptors, they rapidly induce inflammatory responses. This process is tightly controlled at the transcriptional level. The DNA binding zinc-finger protein CCCTC-binding factor (Ctcf) is a crucial regulator of long-range chromatin interactions and coordinates specific communication between transcription factors and gene expression processes. In this study, the Ctcf gene was specifically deleted in myeloid cells by making use of the transgenic Cre-LoxP system. Conditional deletion of the Ctcf gene in myeloid cells induced a mild phenotype in vivo. Ctcf-deficient mice exhibited significantly reduced expression of major histocompatibility complex (MHC) class II in the liver. Ctcf-deficient macrophages demonstrated a normal surface phenotype and phagocytosis capacity. Upon Toll-like receptor (TLR) stimulation, they produced normal levels of the pro-inflammatory cytokines IL-12 and IL-6, but manifested a strongly impaired capacity to produce tumor-necrosis factor (TNF) and IL-10, as well as to express the IL-10 family members IL-19, IL-20 and IL-24. Taken together, our data demonstrate a role of Ctcf that involves fine-tuning of macrophage function.

Gene expression profiling in mice with enforced Gata3 expression reveals putative targets of Gata3 in double positive thymocytes.

The zinc-finger transcription factors Gata3 and ThPOK have both been implicated in positive selection of double positive (DP) thymocytes towards the CD4 lineage. As in the absence of Gata3, expression of ThPOK is lacking, Gata3 may directly regulate ThPOK expression. As ThPOK failed to promote CD4(+) lineage differentiation of Gata3-deficient cells, ThPOK cannot be the only Gata3 target gene essential for the induction of the CD4(+) lineage program. Therefore, it is conceivable that Gata3 is essential for selected DP T cells to reach the developmental stage at which ThPOK expression is induced. Here, we show that Gata3 overexpression does not affect ThPOK expression levels in DP or CD4(+) thymocytes, providing evidence that Gata3 does not directly regulate ThPOK. To identify additional target genes that clarify Gata3 function at the DP thymocyte stage, we performed gene expression profiling assays in wild-type mice and transgenice mice with enforced expression of Gata3, in the presence or absence of the MHC class II-restricted DO11.10 TCR. We found that Gata3 expression in DP cells undergoing positive selection was associated with downregulation of the V(D)J-recombination machinery genes Rag1, Rag2 and TdT. Moreover, Gata3 overexpression was associated with downregulation of many signaling molecules and the induction of modulators of TCR signaling, including Ctla-4 and thrombospondin 2. Together with our previous finding that Gata3 reduces expression of CD5, a negative regulator of TCR signaling, and upregulates TCR expression, these findings indicate that Gata3 in DP cells mainly functions to (i) terminate TCRalpha gene rearrangement, and (ii) regulate TCR signal intensity or duration in cells undergoing positive selection towards the CD4 lineage.

Enforced expression of GATA3 allows differentiation of IL-17-producing cells, but constrains Th17-mediated pathology.

The zinc-finger transcription factor GATA3 serves as a master regulator of T-helper-2 (Th2) differentiation by inducing expression of the Th2 cytokines IL-4, IL-5 and IL-13 and by suppressing Th1 development. Here, we investigated how GATA3 affects Th17 differentiation, using transgenic mice with enforced GATA3 expression. We activated naïve primary T cells in vitro in the presence of transforming growth factor-beta and IL-6, and found that enforced GATA3 expression induced co-expression of Th2 cytokines in IL-17-producing T cells. Although the presence of IL-4 hampered Th17 differentiation, transforming growth factor-beta/IL-6 cultures from GATA3 transgenic mice contained substantial numbers of IL-17(+) cells, partially because GATA3 supported Th17 differentiation by limiting IL-2 and IFN-gamma production. GATA3 additionally constrained Th17 differentiation in vitro through IL-4-independent mechanisms, involving downregulating transcription of STAT3, STAT4, NFATc2 and the nuclear factor RORgammat, which is crucial for Th17 differentiation. Remarkably, upon myelin oligodendrocyte glycoprotein immunization in vivo, GATA3 transgenic mice contained similar numbers of IL-17-producing T cells in their lymph nodes as wild-type mice, but were not susceptible to autoimmune encephalomyelitis, possibly due to concomitant production of IL-4 and IL-10 induction. We therefore conclude that although GATA3 allows Th17 differentiation, it acts as an inhibitor of Th17-mediated pathology, through IL-4-dependent and IL-4-independent pathways.