Sequential Enhancer Sequestration Dysregulates Recombination Center Formation at the IgH Locus.

Immunoglobulin heavy-chain (IgH) genes are assembled by DNA rearrangements that juxtapose a variable (VH), a diversity (DH), and a joining (JH) gene segment. Here, we report that in the absence of intergenic control region 1 (IGCR1), the intronic enhancer (Eµ) associates with the next available CTCF binding site located close to VH81X via putative heterotypic interactions involving YY1 and CTCF. The alternate Eµ/VH81X loop leads to formation of a distorted recombination center and altered DH rearrangements and disrupts chromosome conformation that favors distal VH recombination. Cumulatively, these features drive highly skewed, Eµ-dependent recombination of VH81X. Sequential deletion of CTCF binding regions on IGCR1-deleted alleles suggests that they influence recombination of single proximal VH gene segments. Our observations demonstrate that Eµ interacts differently with IGCR1- or VH-associated CTCF binding sites and thereby identify distinct roles for insulator-like elements in directing enhancer activity.

Localized epigenetic changes induced by DH recombination restricts recombinase to DJH junctions.

Genes encoding immunoglobulin heavy chains (Igh) are assembled by rearrangement of variable (V(H)), diversity (D(H)) and joining (J(H)) gene segments. Three critical constraints govern V(H) recombination. These include timing (V(H) recombination follows D(H) recombination), precision (V(H) gene segments recombine only to DJ(H) junctions) and allele specificity (V(H) recombination is restricted to DJ(H)-recombined alleles). Here we provide a model for these universal features of V(H) recombination. Analyses of DJ(H)-recombined alleles showed that DJ(H) junctions were selectively epigenetically marked, became nuclease sensitive and bound RAG recombinase proteins, which thereby permitted D(H)-associated recombination signal sequences to initiate the second step of Igh gene assembly. We propose that V(H) recombination is precise, because these changes did not extend to germline D(H) segments located 5' of the DJ(H) junction.

T cell factor 1 initiates the T helper type 2 fate by inducing the transcription factor GATA-3 and repressing interferon-gamma.

The differentiation of activated CD4(+) T cells into the T helper type 1 (T(H)1) or T(H)2 fate is regulated by cytokines and the transcription factors T-bet and GATA-3. Whereas interleukin 12 (IL-12) produced by antigen-presenting cells initiates the T(H)1 fate, signals that initiate the T(H)2 fate are not completely characterized. Here we show that early GATA-3 expression, required for T(H)2 differentiation, was induced by T cell factor 1 (TCF-1) and its cofactor beta-catenin, mainly from the proximal Gata3 promoter upstream of exon 1b. This activity was induced after T cell antigen receptor (TCR) stimulation and was independent of IL-4 receptor signaling through the transcription factor STAT6. Furthermore, TCF-1 blocked T(H)1 fate by negatively regulating interferon-gamma (IFN-gamma) expression independently of beta-catenin. Thus, TCF-1 initiates T(H)2 differentiation of activated CD4(+) T cells by promoting GATA-3 expression and suppressing IFN-gamma expression.

A histone-fold complex and FANCM form a conserved DNA-remodeling complex to maintain genome stability.

FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here, we show that FANCM forms a conserved DNA-remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human.

Activation of 12/23-RSS-dependent RAG cleavage by hSWI/SNF complex in the absence of transcription.

Maintenance of genomic integrity during antigen receptor gene rearrangements requires (1) regulated access of the V(D)J recombinase to specific loci and (2) generation of double-strand DNA breaks only after recognition of a pair of matched recombination signal sequences (RSSs). Here we recapitulate both key aspects of regulated recombinase accessibility in a cell-free system using plasmid substrates assembled into chromatin. We show that recruitment of the SWI/SNF chromatin-remodeling complex to both RSSs increases coupled cleavage by RAG1 and RAG2 proteins. SWI/SNF functions by altering local chromatin structure in the absence of RNA polymerase II-dependent transcription or histone modifications. These observations demonstrate a direct role for cis-sequence-regulated local chromatin remodeling in RAG1/2-dependent initiation of V(D)J recombination.

Three-dimensional chromatin reorganization regulates B cell development during ageing.

The contribution of three-dimensional genome organization to physiological ageing is not well known. Here we show that large-scale chromatin reorganization distinguishes young and old bone marrow progenitor (pro-) B cells. These changes result in increased interactions at the compartment level and reduced interactions within topologically associated domains (TADs). The gene encoding Ebf1, a key B cell regulator, switches from compartment A to B with age. Genetically reducing Ebf1 recapitulates some features of old pro-B cells. TADs that are most reduced with age contain genes important for B cell development, including the immunoglobulin heavy chain (Igh) locus. Weaker intra-TAD interactions at Igh correlate with altered variable (V), diversity (D) and joining (J) gene recombination. Our observations implicate three-dimensional chromatin reorganization as a major driver of pro-B cell phenotypes that impair B lymphopoiesis with age.

Using BAC transgenesis in zebrafish to identify regulatory sequences of the amyloid precursor protein gene in humans.

BACKGROUND: Non-coding DNA in and around the human Amyloid Precursor Protein (APP) gene that is central to Alzheimer's disease (AD) shares little sequence similarity with that of appb in zebrafish. Identifying DNA domains regulating expression of the gene in such situations becomes a challenge. Taking advantage of the zebrafish system that allows rapid functional analyses of gene regulatory sequences, we previously showed that two discontinuous DNA domains in zebrafish appb are important for expression of the gene in neurons: an enhancer in intron 1 and sequences 28-31 kb upstream of the gene. Here we identify the putative transcription factor binding sites responsible for this distal cis-acting regulation, and use that information to identify a regulatory region of the human APP gene. RESULTS: Functional analyses of intron 1 enhancer mutations in enhancer-trap BACs expressed as transgenes in zebrafish identified putative binding sites of two known transcription factor proteins, E4BP4/ NFIL3 and Forkhead, to be required for expression of appb. A cluster of three E4BP4 sites at -31 kb is also shown to be essential for neuron-specific expression, suggesting that the dependence of expression on upstream sequences is mediated by these E4BP4 sites. E4BP4/ NFIL3 and XFD1 sites in the intron enhancer and E4BP4/ NFIL3 sites at -31 kb specifically and efficiently bind the corresponding zebrafish proteins in vitro. These sites are statistically over-represented in both the zebrafish appb and the human APP genes, although their locations are different. Remarkably, a cluster of four E4BP4 sites in intron 4 of human APP exists in actively transcribing chromatin in a human neuroblastoma cell-line, SHSY5Y, expressing APP as shown using chromatin immunoprecipitation (ChIP) experiments. Thus although the two genes share little sequence conservation, they appear to share the same regulatory logic and are regulated by a similar set of transcription factors. CONCLUSION: The results suggest that the clock-regulated and immune system modulator transcription factor E4BP4/ NFIL3 likely regulates the expression of both appb in zebrafish and APP in humans. It suggests potential human APP gene regulatory pathways, not on the basis of comparing DNA primary sequences with zebrafish appb but on the model of conservation of transcription factors.

Misregulation of the IgH Locus in Thymocytes.

Functional antigen receptor genes are assembled by somatic rearrangements that are largely lymphocyte lineage specific. The immunoglobulin heavy chain (IgH) gene locus is unique amongst the seven antigen receptor loci in undergoing partial gene rearrangements in the wrong lineage. Here we demonstrate that breakdown of lineage-specificity is associated with inappropriate activation of the Eµ enhancer during T cell development by a different constellation of transcription factors than those used in developing B cells. This is reflected in reduced enhancer-induced epigenetic changes, eRNAs, formation of the RAG1/2-rich recombination center, attenuated chromatin looping and markedly different utilization of DH gene segments in CD4+CD8+ (DP) thymocytes. Additionally, CTCF-dependent VH locus compaction is disrupted in DP cells despite comparable transcription factor binding in both lineages. These observations identify multiple mechanisms that contribute to lineage-specific antigen receptor gene assembly.

Spt5 accumulation at variable genes distinguishes somatic hypermutation in germinal center B cells from ex vivo-activated cells.

Variable (V) genes of immunoglobulins undergo somatic hypermutation by activation-induced deaminase (AID) to generate amino acid substitutions that encode antibodies with increased affinity for antigen. Hypermutation is restricted to germinal center B cells and cannot be recapitulated in ex vivo-activated splenic cells, even though the latter express high levels of AID. This suggests that there is a specific feature of antigen activation in germinal centers that recruits AID to V genes which is absent in mitogen-activated cultured cells. Using two Igh knock-in mouse models, we found that RNA polymerase II accumulates in V regions in B cells after both types of stimulation for an extended distance of 1.2 kb from the TATA box. The paused polymerases generate abundant single-strand DNA targets for AID. However, there is a distinct accumulation of the initiating form of polymerase, along with the transcription cofactor Spt5 and AID, in the V region from germinal center cells, which is totally absent in cultured cells. These data support a model where mutations are prevalent in germinal center cells, but not in ex vivo cells, because the initiating form of polymerase is retained, which affects Spt5 and AID recruitment.

A targeted bypass screen identifies Ynl187p, Prp42p, Snu71p, and Cbp80p for stable U1 snRNP/Pre-mRNA interaction.

To understand how DEXD/H-box proteins recognize and interact with their cellular substrates, we have been studying Prp28p, a DEXD/H-box splicing factor required for switching the U1 snRNP with the U6 snRNP at the precursor mRNA (pre-mRNA) 5' splice site. We previously demonstrated that the requirement for Prp28p can be eliminated by mutations that alter either the U1 snRNA or the U1C protein, suggesting that both are targets of Prp28p. Inspired by this finding, we designed a bypass genetic screen to specifically search for additional, novel targets of Prp28p. The screen identified Prp42p, Snu71p, and Cbp80p, all known components of commitment complexes, as well as Ynl187p, a protein of uncertain function. To examine the role of Ynl187p in splicing, we carried out extensive genetic and biochemical analysis, including chromatin immunoprecipitation. Our data suggest that Ynl187p acts in concert with U1C and Cbp80p to help stabilize the U1 snRNP-5' splice site interaction. These findings are discussed in the context of DEXD/H-box proteins and their role in vivo as well as the potential need for more integral U1-snRNP proteins in governing the fungal 5' splice site RNA-RNA interaction compared to the number of U1 snRNP proteins needed by metazoans.

Mi2beta shows chromatin enzyme specificity by erasing a DNase I-hypersensitive site established by ACF.

ATP-dependent chromatin-remodeling enzymes are linked to changes in gene expression; however, it is not clear how the multiple remodeling enzymes found in eukaryotes differ in function and work together. In this report, we demonstrate that the ATP-dependent remodeling enzymes ACF and Mi2beta can direct consecutive, opposing chromatin-remodeling events, when recruited to chromatin by different transcription factors. In a cell-free system based on the immunoglobulin heavy chain gene enhancer, we show that TFE3 induces a DNase I-hypersensitive site in an ATP-dependent reaction that requires ACF following transcription factor binding to chromatin. In a second step, PU.1 directs Mi2beta to erase an established DNase I-hypersensitive site, in an ATP-dependent reaction subsequent to PU.1 binding to chromatin, whereas ACF will not support erasure. Erasure occurred without displacing the transcription factor that initiated the site. Other tested enzymes were unable to erase the DNase I-hypersensitive site. Establishing and erasing the DNase I-hypersensitive site required transcriptional activation domains from TFE3 and PU.1, respectively. Together, these results provide important new mechanistic insight into the combinatorial control of chromatin structure.

Nucleophosmin/B23 negatively regulates GCN5-dependent histone acetylation and transactivation.

Nucleophosmin/B23 is a multifunctional phosphoprotein that is overexpressed in cancer cells and has been shown to be involved in both positive and negative regulation of transcription. In this study, we first identified GCN5 acetyltransferase as a B23-interacting protein by mass spectrometry, which was then confirmed by in vivo co-immunoprecipitation. An in vitro assay demonstrated that B23 bound the PCAF-N domain of GCN5 and inhibited GCN5-mediated acetylation of both free and mononucleosomal histones, probably through interfering with GCN5 and masking histones from being acetylated. Mitotic B23 exhibited higher inhibitory activity on GCN5-mediated histone acetylation than interphase B23. Immunodepletion experiments of mitotic extracts revealed that phosphorylation of B23 at Thr 199 enhanced the inhibition of GCN5-mediated histone acetylation. Moreover, luciferase reporter and microarray analyses suggested that B23 attenuated GCN5-mediated transactivation in vivo. Taken together, our studies suggest a molecular mechanism of B23 in the mitotic inhibition of GCN5-mediated histone acetylation and transactivation.

The U1 snRNP protein U1C recognizes the 5' splice site in the absence of base pairing.

Splicing of precursor messenger RNA takes place in the spliceosome, a large RNA/protein macromolecular machine. Spliceosome assembly occurs in an ordered pathway in vitro and is conserved between yeast and mammalian systems. The earliest step is commitment complex formation in yeast or E complex formation in mammals, which engages the pre-mRNA in the splicing pathway and involves interactions between U1 small nuclear ribonucleoprotein (snRNP) and the pre-mRNA 5' splice site. Complex formation depends on highly conserved base pairing between the 5' splice site and the 5' end of U1 snRNA, both in vivo and in vitro. U1 snRNP proteins also contribute to U1 snRNP activity. Here we show that U1 snRNP lacking the 5' end of its snRNA retains 5'-splice-site sequence specificity. We also show that recombinant yeast U1C protein, a U1 snRNP protein, selects a 5'-splice-site-like sequence in which the first four nucleotides, GUAU, are identical to the first four nucleotides of the yeast 5'-splice-site consensus sequence. We propose that a U1C 5'-splice-site interaction precedes pre-mRNA/U1 snRNA base pairing and is the earliest step in the splicing pathway.

Effects of the U1C L13 mutation and temperature regulation of yeast commitment complex formation.

The U1 small nuclear ribonucleoprotein particle U1C protein has a zinc finger-like structure (C2H2 motif) at its N terminus, which is conserved from yeast to humans. Mutations of amino acid L13 within this domain rescue the essential function of the helicase protein Prp28p. Prp28p has been implicated in unwinding the 5' splice site (5'ss)-U1 small nuclear RNA (snRNA) base-pairing, to allow replacement of U1 snRNA with U6 snRNA during spliceosome assembly. The L13 phenotype has therefore been interpreted to indicate that WT U1C contributes to 5'ss-U1 snRNA stabilization by binding to the RNA duplex. We show here that an L13 mutant extract cannot form stable base-pairing at room temperature but is permissive for U1-5'ss base-pairing at low temperature. This phenotype is similar to that of a U1C-depleted extract, indicating that the U1C L13 mutation is a strong loss-of-function mutation. The two mutant extracts are unlike a WT extract, which undergoes stable pairing at room temperature but little or no pairing at low temperature. Taken together with previous results and the failure to observe a direct interaction of U1C with the U1-5'ss duplex, the data suggest that U1C contributes indirectly to stable U1-5'ss base-pairing under permissive conditions. A model is proposed to account for the L13 results.