The RNA-binding protein FUS/TLS interacts with SPO11 and PRDM9 and localize at meiotic recombination hotspots.

In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11ß and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.

On the dependent recognition of some long zinc finger proteins.

The human genome contains about 800 C2H2 zinc finger proteins (ZFPs), and most of them are composed of long arrays of zinc fingers. Standard ZFP recognition model asserts longer finger arrays should recognize longer DNA-binding sites. However, recent experimental efforts to identify in vivo ZFP binding sites contradict this assumption, with many exhibiting short motifs. Here we use ZFY, CTCF, ZIM3, and ZNF343 as examples to address three closely related questions: What are the reasons that impede current motif discovery methods? What are the functions of those seemingly unused fingers and how can we improve the motif discovery algorithms based on long ZFPs' biophysical properties? Using ZFY, we employed a variety of methods and find evidence for 'dependent recognition' where downstream fingers can recognize some previously undiscovered motifs only in the presence of an intact core site. For CTCF, high-throughput measurements revealed its upstream specificity profile depends on the strength of its core. Moreover, the binding strength of the upstream site modulates CTCF's sensitivity to different epigenetic modifications within the core, providing new insight into how the previously identified intellectual disability-causing and cancer-related mutant R567W disrupts upstream recognition and deregulates the epigenetic control by CTCF. Our results establish that, because of irregular motif structures, variable spacing and dependent recognition between sub-motifs, the specificities of long ZFPs are significantly underestimated, so we developed an algorithm, ModeMap, to infer the motifs and recognition models of ZIM3 and ZNF343, which facilitates high-confidence identification of specific binding sites, including repeats-derived elements. With revised concept, technique, and algorithm, we can discover the overlooked specificities and functions of those 'extra' fingers, and therefore decipher their broader roles in human biology and diseases.

Gammaherpesvirus-mediated repression reveals EWSR1 to be a negative regulator of B cell responses.

The germinal center (GC) plays a central role in the generation of antigen-specific B cells and antibodies. Tight regulation of the GC is essential due to the inherent risks of tumorigenesis and autoimmunity posed by inappropriate GC B cell processes. Gammaherpesviruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) utilize numerous armaments to drive infected naïve B cells, independent of antigen, through GC reactions to expand the latently infected B cell population and establish a stable latency reservoir. We previously demonstrated that the MHV68 microRNA (miRNA) mghv-miR-M1-7-5p represses host EWSR1 (Ewing sarcoma breakpoint region 1) to promote B cell infection. EWSR1 is a transcription and splicing regulator that is recognized for its involvement as a fusion protein in Ewing sarcoma. A function for EWSR1 in B cell responses has not been previously reported. Here, we demonstrate that 1) B cell-specific deletion of EWSR1 had no effect on generation of mature B cell subsets or basal immunoglobulin levels in naïve mice, 2) repression or ablation of EWSR1 in B cells promoted expansion of MHV68 latently infected GC B cells, and 3) B cell-specific deletion of EWSR1 during a normal immune response to nonviral antigen resulted in significantly elevated numbers of antigen-specific GC B cells, plasma cells, and circulating antibodies. Notably, EWSR1 deficiency did not affect the proliferation or survival of GC B cells but instead resulted in the generation of increased numbers of precursor GC B cells. Cumulatively, these findings demonstrate that EWSR1 is a negative regulator of B cell responses.

Mouse EWSR1 is crucial for spermatid post-meiotic transcription and spermiogenesis.

Spermatogenesis is precisely controlled by complex gene-expression programs. During mammalian male germ-cell development, a crucial feature is the repression of transcription before spermatid elongation. Previously, we discovered that the RNA-binding protein EWSR1 plays an important role in meiotic recombination in mouse, and showed that EWSR1 is highly expressed in late meiotic cells and post-meiotic cells. Here, we used an Ewsr1 pachytene stage-specific knockout mouse model to study the roles of Ewsr1 in late meiotic prophase I and in spermatozoa maturation. We show that loss of EWSR1 in late meiotic prophase I does not affect proper meiosis completion, but does result in defective spermatid elongation and chromocenter formation in the developing germ cells. As a result, male mice lacking EWSR1 after pachynema are sterile. We found that, in Ewsr1 CKO round spermatids, transition from a meiotic gene-expression program to a post-meiotic and spermatid gene expression program related to DNA condensation is impaired, suggesting that EWSR1 plays an important role in regulation of spermiogenesis-related mRNA synthesis necessary for spermatid differentiation into mature sperm.

EWSR1 affects PRDM9-dependent histone 3 methylation and provides a link between recombination hotspots and the chromosome axis protein REC8.

Meiotic recombination in most mammals requires recombination hotspot activation through the action of the histone 3 Lys-4 and Lys-36 methyltransferase PRDM9 to ensure successful double-strand-break initiation and repair. Here we show that EWSR1, a protein whose role in meiosis was not previously clarified in detail, binds to both PRDM9 and pREC8, a phosphorylated meiosis-specific cohesin, in male meiotic cells. We created a Ewsr1 conditional knockout mouse model to deplete EWSR1 before the onset of meiosis and found that absence of EWSR1 causes meiotic arrest with decreased histone trimethylation at meiotic hotspots, impaired DNA double-strand-break repair, and reduced crossover number. Our results demonstrate that EWSR1 is essential for promoting PRDM9-dependent histone methylation and normal meiotic progress, possibly by facilitating the linking between PRDM9-bound hotspots and the nascent chromosome axis through its component cohesin pREC8.

Colocalization of somatic and meiotic double strand breaks near the Myc oncogene on mouse chromosome 15.

Both somatic and meiotic recombinations involve the repair of DNA double strand breaks (DSBs) that occur at preferred locations in the genome. Improper repair of DSBs during either mitosis or meiosis can lead to mutations, chromosomal aberration such as translocations, cancer, and/or cell death. Currently, no model exists that explains the locations of either spontaneous somatic DSBs or programmed meiotic DSBs or relates them to each other. One common class of tumorigenic translocations arising from DSBs is chromosomal rearrangements near the Myc oncogene. Myc translocations have been associated with Burkitt lymphoma in humans, plasmacytoma in mice, and immunocytoma in rats. Comparing the locations of somatic and meiotic DSBs near the mouse Myc oncogene, we demonstrated that the placement of these DSBs is not random and that both events clustered in the same short discrete region of the genome. Our work shows that both somatic and meiotic DSBs tend to occur in proximity to each other within the Myc region, suggesting that they share common originating features. It is likely that some regions of the genome are more susceptible to both somatic and meiotic DSBs, and the locations of meiotic hotspots may be an indicator of genomic regions more susceptible to DNA damage.

Expression and localization of PCSK9 in rat hepatic cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), recently cloned in several laboratories, including ours, causes a third form of autosomal dominant hypercholesterolemia. Its mechanism of action remains unclear. We studied the expression and subcellular localization of PCSK9 in fetal and adult rat tissues associated with cholesterol homeostasis using quantitative reverse transcriptase--PCR, Western blot analysis, subcellular fractionation, and confocal immunofluorescent microscopy. PCSK9 mRNA is most abundant in yolk sac and fetal liver, but the highest expression of the protein was found in differentiated hepatoma FAO-1 cell line, which also shows the highest expression of LDLR. In FAO-1 cells PCSK9 expression is downregulated by cholesterol and 25-hydroxycholesterol and upregulated in the absence of sterols following the same pattern of expression as HMG-CoA reductase, synthase, and LDLR. Subcellular fractionation, combined with Western blotting, showed that PCSK9 is localized in the ER and intermediate vesicular compartment of the cell but not in Golgi cisternae. The mature enzyme is secreted from the liver and hepatoma cells. Double labeling with antibodies to PCSK9 and LDLR or clathrin revealed some colocalization of PCSK9 with clathrin-coated vesicles and LDLR. In conclusion, our results show that PCSK9 is processed in the ER, and the mature convertase is secreted in the plasma.