Pronounced cohabitation of active immunoglobulin genes from three different chromosomes in transcription factories during maximal antibody synthesis.

To understand the relationships between nuclear organization and gene expression in a model system, we employed three-dimensional imaging and chromatin immunoprecipitation (ChIP)-chromosome conformation capture (3C) techniques to investigate the topographies of the immunoglobulin (Ig) genes and transcripts during B-cell development. Remarkably, in plasma cells, when antibody synthesis peaks, active Ig genes residing on three different chromosomes exhibit pronounced colocalizations in transcription factories, often near the nuclear periphery, and display trans-chromosomal enhancer interactions, and their transcripts frequently share interchromatin trafficking channels. Conceptually, these features of nuclear organization maximize coordinated transcriptional and transcript trafficking control for potentiating the optimal cytoplasmic assembly of the resulting translation products into protein multimers.

A major deletion in the Vκ-Jκ intervening region results in hyperelevated transcription of proximal Vκ genes and a severely restricted repertoire.

Our previous studies have shown that DNase I hypersensitive sites 1 and 2 (HS1-2) and HS3-6 within the mouse Vκ-Jκ intervening region are essential for controlling locus contraction and creating a diverse Ab repertoire. In this article, we demonstrate that a 6.3-kb deletion encompassing HS1-6 altogether not only leads to the predictable sums of these phenotypes, but also results in a novel hyperelevation of transcription of proximal Vκ genes, in both pre-B and splenic B cells. These findings reveal previously unrecognized additional functions for cis-elements within the Vκ-Jκ intervening region, namely, prevention of the production of massive levels of noncoding RNA species by silencing transcription of germline proximal Vκ genes in both developing and mature B cells.

Vκ gene repertoire and locus contraction are specified by critical DNase I hypersensitive sites within the Vκ-Jκ intervening region.

The processes of Ig gene locus contraction and looping during V(D)J-recombination are essential for creating a diverse Ab repertoire. However, no cis-acting sequence that plays a major role in specifying locus contraction has been uncovered within the Igκ gene locus. In this article, we demonstrate that a 650-bp sequence corresponding to DNase I hypersensitive sites HS1-2 within the mouse Igκ gene V-J intervening region binds CCCTC-binding factor and specifies locus contraction and long-range Vκ gene usage spanning 3.2 Mb in pre-B cells. We call this novel element Cer (for "contracting element for recombination"). Targeted deletion of Cer caused markedly increased proximal and greatly diminished upstream Vκ gene usage, higher allele usage, more splenic Igκ(+) B cells, and nonlineage-specific Igκ rearrangement in T cells. Relative to wild-type mice, Cer-deletion mice exhibited similar levels of Vκ gene germline transcription and H3K4me3 epigenetic marks but displayed a dramatic decrease in locus contraction in pre-B cells. Thus, our studies demonstrate that DNase I hypersensitive sites HS1-2 within the Vκ-Jκ intervening region are essential for controlling locus contraction and creating a diverse Ab repertoire.

A new hypersensitive site, HS10, and the enhancers, E3' and Ed, differentially regulate Igκ gene expression.

The mouse Igκ gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3' enhancer (E3'), and a further downstream enhancer (Ed). We previously discovered, using the chromosome conformation-capture technique, that Ei and E3' interact with a novel DNA sequence near the 3' end of the Igκ locus, specifically in B cells. In the present investigation, we examined the function of this far downstream element. The sequence is evolutionarily conserved and exhibits a plasmacytoma cell-specific DNase I-hypersensitive site in chromatin, henceforth termed HS10 in the locus. HS10 acts as a coactivator of E3' in transient transfection assays. Although HS10(-/-) mice exhibited normal patterns of B cell development, they were tested further along with E3'(-/-) and Ed(-/-) mice for their Igκ expression levels in plasma cells, as well as for both allelic and isotype exclusion in splenic B cells. HS10(-/-) and Ed(-/-), but not E3'(-/-), mice exhibited 2.5-fold lower levels of Igκ expression in antigenically challenged plasma cells. E3'(-/-) mice, but not HS10(-/-) mice, exhibited impaired IgL isotype and allelic exclusion in splenic B cells. We have suggestive results that Ed may also weakly participate in these processes. In addition, HS10(-/-) mice no longer exhibited regional chromosome interactions with E3', and they exhibited modestly reduced somatic hypermutation in the Jκ-Cκ intronic region in germinal center B cells from Peyer's patches. We conclude that the HS10, E3', and Ed differentially regulate Igκ gene dynamics.

A multifunctional element in the mouse Igκ locus that specifies repertoire and Ig loci subnuclear location.

Nonbiased V gene usage for V(D)J joining is essential for providing an optimal immune system, but no cis-acting sequence with this function has been uncovered. We previously identified a recombination silencer and heterochromatin targeting element in the Vκ-Jκ intervening sequence of germline Igκ transgenes, which we termed Sis. We now have generated Sis knockout mice in the endogenous locus. Intriguingly, Sis(-/-) mice exhibit a skewed Igκ repertoire with markedly decreased distal and enhanced proximal Vκ gene usage for primary rearrangement, which is associated with reduced occupancy of Ikaros and CCCTC-binding factor in the Vκ-Jκ intervening sequence in pre-B cells, proteins believed to be responsible for dampening the recombination of nearby Vκ genes and altering higher-order chromatin looping. Furthermore, monoallelic heterochromatin localization is significantly reduced in Sis(-/-) mice for Igκ in cis and IgH loci in trans in pre-B cells. Because Sis(-/-) mice still allelically excluded Igκ and IgH loci and still exhibited IgL isotype exclusion, we concluded that stable localization at pericentromeric heterochromatin is neither necessary nor sufficient for the establishment or maintenance of allelic exclusion. Hence, Sis is a novel multifunctional element that specifies repertoire and heterochromatin localization to Ig genes.

The Igκ gene enhancers, E3' and Ed, are essential for triggering transcription.

The mouse Igκ gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3' enhancer (E3'), and a further downstream enhancer (Ed). Previous studies on B lymphocytes derived from mutant embryonic stem cells have shown that deletion of either Ei or E3' significantly reduces Igκ gene rearrangement, whereas the combined deletion of both Ei and E3' eliminates such recombination. Furthermore, deletion of either E3' or Ed significantly reduces rearranged Igκ gene transcription. To determine whether the combined presence of both E3' and Ed are essential for Igκ gene expression, we generated homozygous double knockout (DKO) mice with targeted deletions in both elements. Significantly, homozygous DKO mice were unable to generate κ(+) B cells both in bone marrow and the periphery and exhibited surface expression almost exclusively of Igλ-chains, despite the fact that they possessed potentially functional rearranged Igκ genes. Compared with their single-enhancer-deleted counterparts, Igκ loci in homozygous DKO mice exhibited dramatically reduced germline and rearranged gene transcription, lower levels of gene rearrangement and histone H3 acetylation, and markedly increased DNA methylation. This contributed to a partial developmental block at the pre-B cell stage of development. We conclude that the two downstream enhancers are essential in Igκ gene expression and that Ei in homozygous DKO mice is incapable of triggering Igκ gene transcription. Furthermore, these results reveal unexpected compensatory roles for Ed in E3' knockout mice in triggering germline transcription and Vκ gene rearrangements to both Jκ and RS elements.

Divergent roles of RelA and c-Rel in establishing chromosomal loops upon activation of the Igkappa gene.

Precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening DNA, but how trans-acting regulatory proteins work to establish and maintain DNA loops during gene activation remains largely unexplored. LPS-induced transcription of the mouse Igkappa gene in B lymphocytes utilizes three distal enhancers and requires the transcription factor NF-kappaB, whose family members include RelA and c-Rel. Using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that LPS-induced Igkappa gene activation creates chromosomal loops by bridging together all three pairwise interactions between the distal enhancers and RNA polymerase II, the apparent molecular tie for the bases of these loops. RelA and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis, or c-Rel. We have thus identified both essential and nonessential events that establish higher order chromatin reorganization during Igkappa gene activation.

Engineered apoptotic nucleases for chromatin research.

We have created new genomics tools for chromatin research by genetically engineering the human and mouse major apoptotic nucleases that are responsible for internucleosomal DNA cleavage, DNA fragmentation factor (DFF). Normally, in its inactive form, DFF is a heterodimer composed of a 45-kDa chaperone inhibitor subunit (DFF45 or ICAD), and a 40-kDa latent endonuclease subunit (DFF40 or CAD). Upon caspase-3 cleavage of DFF45, DFF40 forms active endonuclease homo-oligomers. Although Saccharomyces cerevisiae lacks DFF, expression of caspase-3 is lethal in this organism, but expression of the highly sequence-specific tobacco etch virus protease (TEVP) is harmless. Therefore, we inserted TEVP cleavage sites immediately downstream of the two caspase-3 cleavage sites within DFF45, generating a novel form of DFF (DFF-T) whose nuclease activity proved to be exclusively under the control of TEVP. We demonstrate that co-expression of TEVP and DFF-T under galactose control results in nucleosomal DNA laddering and cell death in S. cerevisiae. We also created synthetic DFF genes with optimized codons for high-level expression in Eschericia coli or S. cerevisiae. We further demonstrate the excellence of the synthetic gene products for in vitro mapping of the nucleosome positions and hypersensitive sites in specific genes such as the yeast PHO5.

Long-range interactions between three transcriptional enhancers, active Vkappa gene promoters, and a 3' boundary sequence spanning 46 kilobases.

The mouse immunoglobulin kappa (Igkappa) gene contains an intronic enhancer and two enhancers downstream of its transcription unit. Using chromosome conformation capture technology, we demonstrate that rearranged and actively transcribed Igkappa alleles in MPC-11 plasmacytoma cells exhibit mutual interactions over 22 kb between these three enhancers and Vkappa gene promoters. In addition, the 5' region of the active transcription unit exhibits a continuum of interactions with downstream chromatin segments. We also observe interactions between Ei and E3' with 3' boundary sequences 24 kb downstream of Ed, adjacent to a neighboring housekeeping gene. Very similar interactions between the enhancers are also exhibited by normal B cells isolated from mouse splenic tissue but not by germ line transcriptionally inactive alleles of T cells or P815 mastocytoma cells, which exhibit a seemingly linear chromatin organization. These results fit a looping mechanism for enhancer function like in the beta-globin locus and suggest a dynamic modulation of the spatial organization of the active Igkappa locus. Chromatin immunoprecipitation experiments reveal that the interacting Igkappa gene cis-acting sequences are associated with AP-4, E47, and p65NF-kappaB, potential protein candidates that may be responsible for initiating and/or maintaining the formation of these higher-order complexes. However, S107 plasmacytoma cells that lack NF-kappaB still exhibit mutual interactions between the Igkappa gene enhancers.

The kinetics of V-J joining throughout 3.5 megabases of the mouse Ig kappa locus fit a constrained diffusion model of nuclear organization.

To gain insight into the nuclear organization of the mouse Ig kappa locus and how it may relate to the formation of synapses during recombination, we have studied the kinetics of rearrangement of different V kappa gene families to J kappa gene segments in the pre-B cell line, 103bcl2. Remarkably, V kappa gene families separated by more than 3.5 Mb from J kappa gene segments rearranged with nearly identical kinetics to those as close as 18 kb to J kappa gene segments. These results fit a model of nuclear organization in which the entire V kappa J kappa region resides within a single nuclear subcompartment and is capable of exhibiting multiple reversible contacts through diffusion and Brownian motion.

Loss of an Igκ gene enhancer in mature B cells results in rapid gene silencing and partial reversible dedifferentiation.

We address here whether there is cellular memory of a transcriptional enhancer once it has served its purpose to establish an active chromatin state. We have previously shown that the mouse Igκ gene's downstream enhancers, E3' and Ed, are essential but play redundant roles for establishing transcriptional activity in the locus during B cell development. To determine whether these enhancers are also necessary for the maintenance of transcriptional activity, we conditionally deleted E3' in mature B cells that possessed Ed(-/-) alleles. Upon E3' deletion, the locus became rapidly silenced and lost positive histone epigenetic marks, and the mature B cells partially dedifferentiated, induced RAG-1 and -2 along with certain other pro-B cell makers, and then redifferentiated after triggering Igλ gene rearrangements. We conclude that the Igκ gene's downstream enhancers are essential for both the establishment and maintenance of transcriptional activity and that there is no cellular memory of previous transcriptional activity in this locus. Furthermore, upon enhancer loss, the mature B cells unexpectedly underwent reversible retrograde differentiation. This result establishes that receptor editing can occur in mature B cells and raises the possibility that this may provide a tolerance mechanism for eliminating autoreactive B cells in the periphery.

The Downstream Transcriptional Enhancer, Ed, positively regulates mouse Ig kappa gene expression and somatic hypermutation.

The mouse Igkappa locus has three known transcriptional enhancers: the matrix association region/intronic enhancer, the 3' enhancer (E3'), and the further downstream enhancer (Ed). Previous studies have shown that both matrix association region/intronic and E3' enhancers are required for maximal gene rearrangement of the locus, and that E3' is also required for maximal expression and somatic hypermutation (SHM). To functionally elucidate Ed in vivo, we generated knockout mice with a targeted germline deletion of Ed. Ed deleted homozygous mice (Ed-/-) have moderately reduced numbers of Igkappa expressing B cells and correspondingly increased numbers of Iglambda expressing B cells in spleen. Ed-/- mice also have decreased Igkappa mRNA expression in resting and T cell-dependent activated splenic B cells and reduced Igkappa chains in sera. However, our analysis indicates that Igkappa gene rearrangement is normal in Ed-/- mice. In addition, our results show that Ed-/- mice exhibit reduced SHM in the Igkappa gene J-C intronic region in germinal center B cells from Peyer's patches. We conclude that Ed positively regulates Igkappa gene expression and SHM, but not gene rearrangement.

Unique features of the apoptotic endonuclease DFF40/CAD relative to micrococcal nuclease as a structural probe for chromatin.

The gold standard for studies of nucleosomal chromatin structure for the past 30 years has been the enzyme micrococcal nuclease (MNase). During the course of our studies on the elucidation of the mechanism of action of the apoptotic nuclease DNA fragmentation factor-40 / caspase-activated deoxyribonuclease (DFF40/CAD) on naked DNA and chromatin substrates, it became clear that this enzyme is superior in certain respects to MNase for studying several aspects of chromatin structure. Here we review our published results supporting this statement. Relative to MNase, we have found that DFF40/CAD has the following properties: (i) it does not cut within nucleosomes to generate subnucleosomal DNA fragments; (ii) it is more specific for the linker regions between nucleosomes; (iii) it lacks exonuclease activity; (iv) it is specific for double-stranded DNA and makes exclusively double-stranded breaks; and (v) it attacks histone-H1-containing chromatin more efficiently. Taken together, these facts explain why DFF40/CAD generates sharper oligonucleosomal DNA ladders compared with those generated by MNase. We therefore recommend the following uses for DFF40/CAD for chromatin research: nucleosome isolation, chromatin-remodeling assays, repeat length measurements, and nucleosome-positioning assays along specific sequences. Other uses include footprinting assays of transcription factor positions, shearing chromatin for immunopreciptitation experiments (ChIP), and shearing DNA for recombinant DNA library preparation or for shotgun cloning for sequencing.

The apoptotic endonuclease DFF40/CAD is inhibited by RNA, heparin and other polyanions.

DFF40/CAD, the major apoptotic nuclease, is specific for double-stranded DNA. However, RNA and single-stranded DNA, though not substrates for the enzyme, compete with double-stranded DNA and inhibit its cleavage by the nuclease. In addition, other anionic polymers, like poly-glutamic acid and heparin also inhibit DFF40/CAD, the latter one being highly effective at nanomolar concentrations. The inhibitory poly-anions bind to the nuclease and impair its ability to bind double-stranded DNA. We propose that such poly-anions bind to the positively charged surface formed by alpha4 helices of the DFF40/CAD homodimer. This surface has been proposed recently to bind to either the major groove of DNA or poly (ADP-ribose), another inhibitor of the nuclease.

A recombination silencer that specifies heterochromatin positioning and ikaros association in the immunoglobulin kappa locus.

Allelic exclusion ensures that individual B lymphocytes produce only one kind of antibody molecule. Previous studies have shown that allelic exclusion of the mouse Igkappa locus occurs by the combination of monoallelic silencing and a low level of monoallelic activation for rearrangement combined with a negative feedback loop blocking additional functional rearrangements. Using yeast artificial chromosome-based single-copy isotransgenic mice, we have identified a cis-acting element that negatively regulates rearrangement in this locus, specifically in B cells. The element, termed Sis, resides in the V-J intervening sequence. Sis specifies the targeting of Igkappa transgenes in pre-B and B cells to centromeric heterochromatin and associates with Ikaros, a repressor protein that also colocalizes with centromeric heterochromatin. Significantly, these are hallmarks of silenced endogenous germline Igkappa genes in B cells. These results lead us to propose that Sis participates in the monoallelic silencing aspect of allelic exclusion regulation.

Discovery, regulation, and action of the major apoptotic nucleases DFF40/CAD and endonuclease G.

Toward the end of the 20th and beginning of the 21st centuries, clever in vitro biochemical complementation experiments and genetic screens from the laboratories of Xiaodong Wang, Shigekazu Nagata, and Ding Xue led to the discovery of two major apoptotic nucleases, termed DNA fragmentation factor (DFF) or caspase-activated DNase (CAD) and endonuclease G (Endo G). Both endonucleases attack chromatin to yield 3'-hydroxyl groups and 5'-phosphate residues, first at the level of 50-300 kb cleavage products and next at the level of internucleosomal DNA fragmentation, but these nucleases possess completely different cellular locations in normal cells and are regulated in vastly different ways. In non-apoptotic cells, DFF exists in the nucleus as a heterodimer, composed of a 45 kD chaperone and inhibitor subunit (DFF45) [also called inhibitor of CAD (ICAD-L)] and a 40 kD latent nuclease subunit (DFF40/CAD). Apoptotic activation of caspase-3 or -7 results in the cleavage of DFF45/ICAD and release of active DFF40/CAD nuclease. DFF40's nuclease activity is further activated by specific chromosomal proteins, such as histone H1, HMGB1/2, and topoisomerase II. DFF is regulated by multiple pre- and post-activation fail-safe steps, which include the requirements for DFF45/ICAD, Hsp70, and Hsp40 proteins to mediate appropriate folding during translation to generate a potentially activatable nuclease, and the synthesis in stoichiometric excess of the inhibitors (DFF45/35; ICAD-S/L). By contrast, Endo G resides in the mitochondrial intermembrane space in normal cells, and is released into the nucleus upon apoptotic disruption of mitochondrial membrane permeability in association with co-activators such as apoptosis-inducing factor (AIF). Understanding further regulatory check-points involved in safeguarding non-apoptotic cells against accidental activation of these nucleases remain as future challenges, as well as designing ways to selectively activate these nucleases in tumor cells.

The histone H1 C-terminal domain binds to the apoptotic nuclease, DNA fragmentation factor (DFF40/CAD) and stimulates DNA cleavage.

The apoptotic nuclease, DNA fragmentation factor (DFF40/CAD), is primarily responsible for internucleosomal DNA cleavage during the terminal stages of programmed cell death. Previously, we demonstrated that histone H1 greatly stimulates naked DNA cleavage by this nuclease. Here, we investigate the mechanism of this stimulation with native and recombinant mouse and human histone H1 species. Using a series of truncation mutants of recombinant histone H1-0, we demonstrate that the H1 C-terminal domain (CTD) is responsible for activation of DFF40/CAD. We show further that the intact histone H1-0 CTD and certain synthetic CTD fragments bind to DFF40/CAD and confer upon it an increased ability to bind to DNA. Interestingly, we find that each of the six somatic cell histone H1 isoforms, whose CTDs differ significantly in primary sequence but not amino acid composition, equally activate DFF40/CAD. We conclude that the interactions identified here between the histone H1 CTD and DFF40/CAD target and activate linker DNA cleavage during the terminal stages of apoptosis.

Assembly and analysis of the mouse immunoglobulin kappa gene sequence.

The mechanisms regulating V gene usage leading to the immunoglobulin (Ig) repertoire have been of interest for many years but are only partially defined. To gain insight into these processes, we have assembled the nucleotide sequence of the Mus musculus Igkappa locus using data recently made available from genome-wide sequencing efforts. We found the locus to be 3.21 Mb in length and mapped all known functional, pseudo- and relic V gene segments onto the sequence, along with known regulatory elements. We corrected errors in former gene assignments, positions and orientations and identified a novel Vkappa4 gene segment. This assembly allowed the establishment of a unified nomenclature for the V genes based on their relative positions similar to the nomenclature system adopted for the human Ig loci. The 5' boundary of the locus is defined by the presence of the tumor-associated calcium-signal transducer-2 gene located 19 kb upstream of Vkappa24-140, the most distal V gene. No non- Vkappa genes were found in the sequence of the locus. Detailed analysis of the sequences 0.5 kb upstream, within, and 0.5 kb downstream of each potentially functional V gene revealed interesting patterns of statistically significant clustering of transcription factor consensus binding sites, generally specific to a particular family. We found E boxes were clustered not only in promoter regions, but also nearby recombination signal sequences. Family members of Vkappa4/5 genes exhibit a conserved pattern of octamer sites in their downstream regions, as well as Ebf sites in their introns, and Lef-1 sites in their upstream regions. We discuss potential functional implications of these findings in the context of possible combinatorial mechanisms for targeting V genes for rearrangement. The assembled sequence and its analyses are available as a resource to the scientific community.

Modeling apoptotic chromatin condensation in normal cell nuclei. Requirement for intranuclear mobility and actin involvement.

Hallmarks of the terminal stages of apoptosis are genomic DNA fragmentation and chromatin condensation. Here, we have studied the mechanism of condensation both in vitro and in vivo. We found that DNA fragmentation per se of isolated nuclei from non-apoptotic cells induced chromatin condensation that closely resembles the morphology seen in apoptotic cells, independent of ATP utilization, at physiological ionic strengths. Interestingly, chromatin condensation was accompanied by release of nuclear actin, and both condensation and actin release could be blocked by reversibly pretreating nuclei with Ca2+, Cu2+, diamide, or low pH, procedures shown to stabilize internal nuclear components. Moreover, specific inhibition of nuclear F-actin depolymerization or promotion of its formation also reduced chromatin condensation. Chromatin condensation could also be inhibited by exposing nuclei to reagents that bind to the DNA minor groove, disrupting native nucleosomal DNA wrapping. In addition, in cultured cells undergoing apoptosis, drugs that inhibit depolymerization of actin or bind to the minor groove also reduced chromatin condensation, but not DNA fragmentation. Therefore, the ability of chromatin fragments with intact nucleosomes to form large clumps of condensed chromatin during apoptosis requires the apparent disassembly of internal nuclear structures that may normally constrain chromosome subdomains in non-apoptotic cells.

Subunit structures and stoichiometries of human DNA fragmentation factor proteins before and after induction of apoptosis.

DNA fragmentation factor (DFF) is one of the major endonucleases responsible for internucleosomal DNA cleavage during apoptosis. Understanding the regulatory checkpoints involved in safeguarding non-apoptotic cells against accidental activation of this nuclease is as important as elucidating its activation mechanisms during apoptosis. Here we address these issues by determining DFF native subunit structures and stoichiometries in human cells before and after induction of apoptosis using the technique of native pore-exclusion limit electrophoresis in combination with Western analyses. For comparison, we employed similar techniques with recombinant proteins in conjunction with atomic force microscopy. Before induction of apoptosis, the expression of DFF subunits varied widely among the cell types studied, and the chaperone/inhibitor subunits DFF45 and DFF35 unexpectedly existed primarily as monomers in vast excess of the latent nuclease subunit, DFF40, which was stoichiometrically associated with DFF45 to form heterodimers. DFF35 was exclusively cytoplasmic as a monomer. Nuclease activation upon caspase-3 cleavage of DFF45/DFF35 was accompanied by DFF40 homo-oligomer formation, with a tetramer being the smallest unit. Interestingly, intact DFF45 can inhibit nuclease activity by associating with these homo-oligomers without mediating their disassembly. We conclude that DFF nuclease is regulated by multiple pre- and post-activation fail-safe steps.