Danae Schulz

CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells

Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all VH genes to be brought in proximity with DH-JH segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ~60 sites throughout the VH region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5′ of DFL16 and the 3′ regulatory region, and also the intronic enhancer (Eμ), creating a DH-JH-Eμ-CH domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the DH region and parts of the VH locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.

Chromosomal reinsertion of broken RSS ends during T cell development

The V(D)J recombinase catalyzes DNA transposition and translocation both in vitro and in vivo. Because lymphoid malignancies contain chromosomal translocations involving antigen receptor and protooncogene loci, it is critical to understand the types of “mistakes” made by the recombinase. Using a newly devised assay, we characterized 48 unique TCRβ recombination signal sequence (RSS) end insertions in murine thymocyte and splenocyte genomic DNA samples. Nearly half of these events targeted “cryptic” RSS-like elements. In no instance did we detect target-site duplications, which is a hallmark of recombinase-mediated transposition in vitro. Rather, these insertions were most likely caused by either V(D)J recombination between a bona fide RSS and a cryptic RSS or the insertion of signal circles into chromosomal loci via a V(D)J recombination-like mechanism. Although wild-type, p53, p53 x scid, H2Ax, and ATM mutant thymocytes all showed similar levels of RSS end insertions, core-RAG2 mutant thymocytes showed a sevenfold greater frequency of such events. Thus, the noncore domain of RAG2 serves to limit the extent to which the integrity of the genome is threatened by mistargeting of V(D)J recombination.

Gfi1b negatively regulates Rag expression directly and via the repression of FoxO1.

Gfi1b negatively regulates Rag expression through direct binding to the Rag locus and through inhibition of Foxo1; mice lacking both Gfi1b and Gfi1 exhibit a block in B cell development.

Bromodomain Proteins Contribute to Maintenance of Bloodstream Form Stage Identity in the African Trypanosome

Trypanosoma brucei, the causative agent of African sleeping sickness, is transmitted to its mammalian host by the tsetse. In the fly, the parasite’s surface is covered with invariant procyclin, while in the mammal it resides extracellularly in its bloodstream form (BF) and is densely covered with highly immunogenic Variant Surface Glycoprotein (VSG). In the BF, the parasite varies this highly immunogenic surface VSG using a repertoire of ~2500 distinct VSG genes. Recent reports in mammalian systems point to a role for histone acetyl-lysine recognizing bromodomain proteins in the maintenance of stem cell fate, leading us to hypothesize that bromodomain proteins may maintain the BF cell fate in trypanosomes. Using small-molecule inhibitors and genetic mutants for individual bromodomain proteins, we performed RNA-seq experiments that revealed changes in the transcriptome similar to those seen in cells differentiating from the BF to the insect stage. This was recapitulated at the protein level by the appearance of insect-stage proteins on the cell surface. Furthermore, bromodomain inhibition disrupts two major BF-specific immune evasion mechanisms that trypanosomes harness to evade mammalian host antibody responses. First, monoallelic expression of the antigenically varied VSG is disrupted. Second, rapid internalization of antibodies bound to VSG on the surface of the trypanosome is blocked. Thus, our studies reveal a role for trypanosome bromodomain proteins in maintaining bloodstream stage identity and immune evasion. Importantly, bromodomain inhibition leads to a decrease in virulence in a mouse model of infection, establishing these proteins as potential therapeutic drug targets for trypanosomiasis. Our 1.25Å resolution crystal structure of a trypanosome bromodomain in complex with I-BET151 reveals a novel binding mode of the inhibitor, which serves as a promising starting point for rational drug design.

Base J and H3.V Regulate Transcriptional Termination in Trypanosoma brucei.

Trypanosoma brucei is a protozoan parasite that lacks many transcription factors found in other eukaryotes, such as those whose binding demarcates enhancers. T. brucei retains histone variants and modifications, however, and it is hypothesized that it relies on epigenetic marks to define transcription-related boundaries. The histone H3 variant (H3.V) and an alternate nucleotide, base J (ß-D-glucosyl-hydroxymethyluracil), are two chromatin marks found at both transcription termination sites (TTSs) and telomeres. Here, we report that the absence of both base J and H3.V result in transcription readthrough and the appearance of antisense transcripts near TTSs. Additionally, we find that maintaining the transcriptional silencing of pol I-transcribed telomeric Variant Surface Glycoprotein (VSG) genes appears to be dependent on deposition of H3.V alone. Our study reveals that gene expression depends on different epigenetic cues depending on chromosomal location and on the transcribing polymerase. This work provides insight into how these signals may have evolved into the more nuanced and fine-tuned gene regulatory mechanisms observed in other model systems.

Vesicles as Vehicles for Virulence

Parasites have long been known to influence host responses to infection through the secretion of virulence factors. Extracellular vesicles are emerging as important mediators of these manipulations, and a new study by Szempruch et al. suggests they could play a crucial role in host responses to African trypanosome infections.

A histone code for regulating V(D)J recombination.

In a recent issue of Molecular Cell, Shimazaki et al. (2009) show that an interaction between RAG2 and a methylated histone might play a critical regulatory role in V(D)J recombination by enhancing DNA binding and enzymatic activity of the V(D)J recombinase.

Gfi1 and Gfi1b Repress Rag Transcription in Plasmacytoid Dendritic Cells In Vitro

Growth factor independence genes (Gfi1 and Gfi1b) repress recombination activating genes (Rag) transcription in developing B lymphocytes. Because all blood lineages originate from hematopoietic stem cells (HSCs) and different lineage progenitors have been shown to share transcription factor networks prior to cell fate commitment, we hypothesized that GFI family proteins may also play a role in repressing Rag transcription or a global lymphoid transcriptional program in other blood lineages. We tested the level of Rag transcription in various blood cells when Gfi1 and Gfi1b were deleted, and observed an upregulation of Rag expression in plasmacytoid dendritic cells (pDCs). Using microarray analysis, we observed that Gfi1 and Gfi1b do not regulate a lymphoid or pDC-specific transcriptional program. This study establishes a role for Gfi1 and Gfi1b in Rag regulation in a non-B lineage cell type.

The VEXing problem of monoallelic expression in the African trypanosome

The African trypanosome, Trypanosoma brucei , is a causative agent of African Trypanosomiasis (also known as “sleeping sickness” in humans and “nagana” in cattle) and imposes an enormous economic burden in regions of Sub-Saharan Africa. T. brucei is transmitted to the mammalian host through the bite of the tsetse fly. In the mammal, it survives extracellularly in the bloodstream, eventually migrating into the central nervous system and causing coma and death. To survive in the bloodstream of its mammalian host, T. brucei must evade the host immune system. The parasite is covered by a dense, variant surface glycoprotein (VSG) coat that shields other epitopes on the cell surface from antibodies produced by the host immune system (reviewed in refs. 1 and 2). A strong antibody response is mounted against the VSG upon entry of the parasite into the bloodstream. However, the parasite harbors thousands of variants of the VSG gene and periodically switches the particular variant that is expressed in a process called antigenic variation (known colloquially in T. brucei as “switching”) (1). VSG genes are transcribed from one of ∼15 telomeric bloodstream expression sites (BESs), only one of which is transcriptionally active at any given time. Transcription of VSG genes occurs within a discrete nuclear structure, called the expression site body (ESB) and is driven by Pol I (1). Switching from expression of one VSG to a new VSG can occur by multiple mechanisms, including transcriptional activation of a new BES and recombinatorial mechanisms (1). Analysis of T. brucei populations in mouse models of infection has revealed that a population of trypanosomes can contain parasites expressing as many as 66 different VSGs at any given time (3). However, although the repertoire of VSGs expressed in a population of trypanosomes is quite diverse, a single trypanosome expresses just one … [↵][1]1To whom correspondence may be addressed. Email: dschulz{at}hmc.edu or papavas{at}rockefeller.edu. [1]: #xref-corresp-1-1