Variegated Expression Research Articles

Sequences within and upstream of the mouse Ets1 gene drive high level expression in B cells, but are not sufficient for consistent expression in T cells.

The levels of transcription factor Ets1 are high in resting B and T cells, but are downregulated by signaling through antigen receptors and Toll-like receptors (TLRs). Loss of Ets1 in mice leads to excessive immune cell activation and development of an autoimmune syndrome and reduced Ets1 expression has been observed in human PBMCs in the context of autoimmune diseases. In B cells, Ets1 serves to prevent premature activation and differentiation to antibody-secreting cells. Given these important roles for Ets1 in the immune response, stringent control of Ets1 gene expression levels is required for homeostasis. However, the genetic regulatory elements that control expression of the Ets1 gene remain relatively unknown. Here we identify a topologically-associating domain (TAD) in the chromatin of B cells that includes the mouse Ets1 gene locus and describe an interaction hub that extends over 100 kb upstream and into the gene body. Additionally, we compile epigenetic datasets to find several putative regulatory elements within the interaction hub by identifying regions of high DNA accessibility and enrichment of active enhancer histone marks. Using reporter constructs, we determine that DNA sequences within this interaction hub are sufficient to direct reporter gene expression in lymphoid tissues of transgenic mice. Further analysis indicates that the reporter construct drives faithful expression of the reporter gene in mouse B cells, but variegated expression in T cells, suggesting the existence of T cell regulatory elements outside this region. To investigate how the downregulation of Ets1 transcription is associated with alterations in the epigenetic landscape of stimulated B cells, we performed ATAC-seq in resting and BCR-stimulated primary B cells and identified four regions within and upstream of the Ets1 locus that undergo changes in chromatin accessibility that correlate to Ets1 gene expression. Interestingly, functional analysis of several putative Ets1 regulatory elements using luciferase constructs suggested a high level of functional redundancy. Taken together our studies reveal a complex network of regulatory elements and transcription factors that coordinate the B cell-specific expression of Ets1.

CCAAT Promoter element regulates transgenerational expression of the MHC class I gene

Transgenerational gene expression depends on both underlying DNA sequences and epigenetic modifications. The latter, which can result in transmission of variegated gene expression patterns across multiple generations without DNA alterations, has been termed epigenetic inheritance and has been documented in plants, worms, flies and mammals. Whereas transcription factors binding to cognate DNA sequence elements regulate gene expression, the molecular basis for epigenetic inheritance has been linked to histone and DNA modifications and non-coding RNA. Here we report that mutation of the CCAAT box promoter element abrogates NF-Y binding and disrupts the stable transgenerational expression of an MHC class I transgene. Transgenic mice with a mutated CCAAT box in the MHC class I transgene display variegated expression of the transgene among littermates and progeny in multiple independently derived transgenic lines. After 4 generations, CCAAT mutant transgenic lines derived from a single founder stably displayed distinct patterns of expression. Histone modifications and RNA polymerase II binding correlate with expression of CCAAT mutant transgenic lines, whereas DNA methylation and nucleosome occupancy do not. Mutation of the CCAAT box also results in changes to CTCF binding and DNA looping patterns across the transgene that correlate with expression status. These studies identify the CCAAT promoter element as a regulator of stable transgenerational gene expression such that mutation of the CCAAT box results in variegated transgenerational inheritance. Considering that the CCAAT box is present in 30% of eukaryotic promoters, this study provides insights into how fidelity of gene expression patterns is maintained through multiple generations.

Extrinsic and intrinsic drivers of natural killer cell clonality.

Clonal expansion of antigen-specific lymphocytes is the fundamental mechanism enabling potent adaptive immune responses and the generation of immune memory. Accompanied by pronounced epigenetic remodeling, the massive proliferation of individual cells generates a critical mass of effectors for the control of acute infections, as well as a pool of memory cells protecting against future pathogen encounters. Classically associated with the adaptive immune system, recent work has demonstrated that innate immune memory to human cytomegalovirus (CMV) infection is stably maintained as large clonal expansions of natural killer (NK) cells, raising questions on the mechanisms for clonal selection and expansion in the absence of re-arranged antigen receptors. Here, we discuss clonal NK cell memory in the context of the mechanisms underlying clonal competition of adaptive lymphocytes and propose alternative selection mechanisms that might decide on the clonal success of their innate counterparts. We propose that the integration of external cues with cell-intrinsic sources of heterogeneity, such as variegated receptor expression, transcriptional states, and somatic variants, compose a bottleneck for clonal selection, contributing to the large size of memory NK cell clones.

Variegate expression of Cre recombinase in hematopoietic cells in CD11c-cre transgenic mice

In this study, we performed an in-depth analysis of Cre expression in the widely used CD11c-Cre transgenic mice generated by the group of Boris Reizis. In contrast to previous observation, using the highly sensitive Rosa-26-floxed-tdTomato reporter mouse line, we show variegated expression of Cre in multiple hematopoietic linage cells starting in hematopoietic stem cells. Indeed, we found that in the CD11c-Cre driver mice, Cre is expressed in cDC linage cells and pDC starting from the myeloid dendritic cell precursor, as expected, but also in a substantial fraction of hematopoietic stem cells and common lymphoid progenitors and, consequently, in >50% of all leukocytes. Hence, this study indicates that the reporter mice used to characterize Cre expression in Cre-driver mice should be selected with caution and considering the sensitivity of the reporter system. This study also suggests that the interpretation of some reports using this CD11c-Cre transgenic mice may need to be re-considered based on a careful evaluation of the cell type-specificity of Cre-mediated in their model.

The P. falciparum alternative histones Pf H2A.Z and Pf H2B.Z are dynamically acetylated and antagonized by PfSir2 histone deacetylases at heterochromatin boundaries.

The malaria parasite Plasmodium falciparum relies on variant expression of members of multi-gene families as a strategy for environmental adaptation to promote parasite survival and pathogenesis. These genes are located in transcriptionally silenced DNA regions. A limited number of these genes escape gene silencing, and switching between them confers variant fitness on parasite progeny. Here, we show that PfSir2 histone deacetylases antagonize DNA-interacting acetylated alternative histones at the boundaries between active and silent DNA. This finding implicates acetylated alternative histones in the mechanism regulating P. falciparum variant gene silencing and thus malaria pathogenesis. This work also revealed that acetylation of alternative histones at promoters is dynamically associated with promoter activity across the genome, implicating acetylation of alternative histones in gene regulation genome wide. Understanding mechanisms of gene regulation in P. falciparum may aid in the development of new therapeutic strategies for malaria, which killed 619,000 people in 2021.

The KIR2DL1 intermediate upstream element participates in gene activation

The human KIR genes encode a family of class I MHC receptors that are expressed on subsets of NK cells. The expression of KIR proteins is controlled by a stochastic process, and competition between sense and antisense promoter elements has been suggested to program the variegated expression of these genes. Previous studies have demonstrated distinct roles of distal, intermediate, and proximal sense promoter/enhancer elements in gene activation and expression. Conversely, proximal and intronic antisense promoter transcripts have been associated with gene silencing at different stages of NK cell development. In the current study, we examine the effect of intermediate promoter deletion on KIR2DL1 expression in the YTS cell line. Homozygous deletion of the KIR2DL1 intermediate element did not affect proximal promoter activity but resulted in increased detection of upstream transcripts. No significant changes in alternative mRNA splicing or expression levels of KIR2DL1 protein were observed. However, intermediate element deletion was associated with a reduced frequency of gene activation by 5-azacytidine. Taken together, these results indicate that the intermediate element is not an enhancer required for KIR expression; however, it is required for the efficient activation of the gene.

An inference model gives insights into innate immune adaptation and repertoire diversity

The innate immune system is the body's first line of defense against infection. Natural killer (NK) cells, a vital part of the innate immune system, help to control infection and eliminate cancer. Studies have identified a vast array of receptors that NK cells use to discriminate between healthy and unhealthy cells. However, at present, it is difficult to explain how NK cells will respond to novel stimuli in different environments. In addition, the expression of different receptors on individual NK cells is highly stochastic, but the reason for these variegated expression patterns is unclear. Here, we studied the recognition of unhealthy target cells as an inference problem, where NK cells must distinguish between healthy targets with normal variability in ligand expression and ones that are clear "outliers." Our mathematical model fits well with experimental data, including NK cells' adaptation to changing environments and responses to different target cells. Furthermore, we find that stochastic, "sparse" receptor expression profiles are best able to detect a variety of possible threats, in agreement with experimental studies of the NK cell repertoire. While our study was specifically motivated by NK cells, our model is general and could also apply more broadly to explain principles of target recognition for other immune cell types.

Multivariate analysis of variegated expression in Neurons: A strategy for unbiased localization of gene function to candidate brain regions in larval zebrafish.

Behavioral screens in model organisms have greatly facilitated the identification of genes and genetic pathways that regulate defined behaviors. Identifying the neural circuitry via which specific genes function to modify behavior remains a significant challenge in the field. Tissue- and cell type-specific knockout, knockdown, and rescue experiments serve this purpose, yet in zebrafish screening through dozens of candidate cell-type-specific and brain-region specific driver lines for their ability to rescue a mutant phenotype remains a bottleneck. Here we report on an alternative strategy that takes advantage of the variegation often present in Gal4-driven UAS lines to express a rescue construct in a neuronal tissue-specific and variegated manner. We developed and validated a computational pipeline that identifies specific brain regions where expression levels of the variegated rescue construct correlate with rescue of a mutant phenotype, indicating that gene expression levels in these regions may causally influence behavior. We termed this unbiased correlative approach Multivariate Analysis of Variegated Expression in Neurons (MAVEN). The MAVEN strategy advances the user's capacity to quickly identify candidate brain regions where gene function may be relevant to a behavioral phenotype. This allows the user to skip or greatly reduce screening for rescue and proceed to experimental validation of candidate brain regions via genetically targeted approaches. MAVEN thus facilitates identification of brain regions in which specific genes function to regulate larval zebrafish behavior.

γδ TCRs Function as Innate-like Receptors in the Bovine γδ T Cell Response against Leptospira.

Leptospira serovar Hardjo are bacterial pathogens of cattle that also cause zoonotic disease in humans. Vaccine-mediated protection against Leptospira serovar Hardjo in cattle is associated with a workshop cluster 1 (WC1)+ γδ T cell response that can be recalled in vitro from PBMC by antigenic stimulation. This provides a model system in which to examine protective vaccine-induced γδ T cell responses in a γδ T cell high species. Only a small proportion (5-10%) of WC1+ γδ T cells from immunized cattle are Leptospira responders, implying that Ag specificity is determined by clonally distributed receptors. Both WC1 and TCR are known to be required for Leptospira-specific responses by bovine WC1+ γδ T cells. Through variegated expression patterns and V(D)J recombination, respectively, they have the capacity to confer Ag specificity. In this study, we develop and use a high-throughput TCR-sequencing approach to study the TCRγ and TCRδ repertoires of naive ex vivo PBMC, Leptospira-responding, and Leptospira nonresponding WC1+ γδ T cells to examine the potential role of γδ TCR in determining Ag specificity. Our results provide novel insights into the PBMC γδ TCR repertoires in cattle, demonstrating the TCRγ repertoire to be clonally stratified and essentially public, whereas the TCRδ repertoire shows much higher levels of clonal diversity and is essentially private. TCR repertoire analysis of Leptospira-responding WC1+ γδ T cells identifies no signature of TCR-mediated selection, suggesting that TCR functions largely as an innate-like receptor and does not act as a primary determinant of Ag specificity in the response to this pathogen.

Variation in pH gradients and FLO11 expression in mat biofilms from environmental isolates of the yeast Saccharomyces cerevisiae.

Saccharomyces cerevisiae produces a multicellular phenotype, known as a mat, on a semi‐solid medium. This biofilm phenotype was first described in the lab strain Σ1278b and has been analyzed mostly in this same background. Yeast cells form a mat by spreading across the medium and adhering to each other and the surface, in part through the variegated expression of the cell adhesion, FLO11. This process creates a characteristic floral pattern and generates pH and glucose gradients outward from the center of the mat. Mats are encapsulated in a liquid which may aid in surface spreading and diffusion. Here, we examine thirteen environmental isolates that vary visually in the phenotype. We predicted that mat properties were universal and increased morphological complexity would be associated with more extreme trait values. Our results showed that pH varied significantly among strains, but was not correlated to mat complexity. Only two isolates generated significant liquid boundaries and neither produced visually complex mats. In five isolates, we tracked the initiation of FLO11 using green fluorescent protein (GFP) under the control of the endogenous promoter. Strains varied in when and how much GFP was detected, with increased signal associated with increased morphological complexity. Generally, the signal was strongest in the center of the mat and absent at the expanding edge. Our results show that traits discovered in one background vary and exist independently of mat complexity in natural isolates. The environment may favor different sets of traits, which could have implications for how this yeast adapts to its many ecological niches.

Comparative genetics of KIR haplotype diversity in humans and rhesus macaques: the balancing act.

The role of natural killer (NK) cells is tightly modulated by interactions of killer cell immunoglobulin-like receptors (KIR) with their ligands of the MHC class I family. Several characteristics of the KIR gene products are conserved in primate evolution, like the receptor structures and the variegated expression pattern. At the genomic level, however, the clusters encoding the KIR family display species-specific diversity, reflected by differential gene expansions and haplotype architecture. The human KIR cluster is extensively studied in large cohorts from various populations, which revealed two KIR haplotype groups, A and B, that represent more inhibitory and more activating functional profiles, respectively. So far, genomic KIR analyses in large outbred populations of non-human primate species are lacking. In this study, we roughly quadrupled the number of rhesus macaques studied for their KIR transcriptome (n = 298). Using segregation analysis, we defined 112 unique KIR region configurations, half of which display a more inhibitory profile, whereas the other half has a more activating potential. The frequencies and functional potential of these profiles might mirror the human KIR haplotype groups. However, whereas the human group A and B KIR haplotypes are confined to largely fixed organizations, the haplotypes in macaques feature highly variable gene content. Moreover, KIR homozygosity was hardly encountered in this panel of macaques. This study exhibits highly diverse haplotype architectures in humans and macaques, which nevertheless might have an equivalent effect on the modulation of NK cell activity.

A Posttranscriptional Pathway of CD40 Ligand mRNA Stability Is Required for the Development of an Optimal Humoral Immune Response

CD40 ligand (CD40L) mRNA stability is dependent on an activation-induced pathway that is mediated by the binding complexes containing the multifunctional RNA-binding protein, polypyrimidine tract-binding protein 1 (PTBP1) to a 3' untranslated region of the transcript. To understand the relationship between regulated CD40L and the requirement for variegated expression during a T-dependent response, we engineered a mouse lacking the CD40L stability element (CD40LΔ5) and asked how this mutation altered multiple aspects of the humoral immunity. We found that CD40LΔ5 mice expressed CD40L at 60% wildtype levels, and lowered expression corresponded to significantly decreased levels of T-dependent Abs, loss of germinal center (GC) B cells and a disorganized GC structure. Gene expression analysis of B cells from CD40LΔ5 mice revealed that genes associated with cell cycle and DNA replication were significantly downregulated and genes linked to apoptosis upregulated. Importantly, somatic hypermutation was relatively unaffected although the number of cells expressing high-affinity Abs was greatly reduced. Additionally, a significant loss of plasmablasts and early memory B cell precursors as a percentage of total GL7+ B cells was observed, indicating that differentiation cues leading to the development of post-GC subsets was highly dependent on a threshold level of CD40L. Thus, regulated mRNA stability plays an integral role in the optimization of humoral immunity by allowing for a dynamic level of CD40L expression on CD4 T cells that results in the proliferation and differentiation of pre-GC and GC B cells into functional subsets.

High-Dimensional Single-Cell Proteomic Analysis Reveals Therapeutic Vulnerabilities in Relapsed/Refractory AML By Concomitant Analysis of Cell Surface Antigens, Signaling Pathways and Anti-Apoptotic Proteins

Background: The heterogeneous and adaptive nature of AML-associated genomic and proteomic landscape may account for disease relapse and poor prognosis, as therapy-associated selective pressure drives the emergence and expansion of AML clones with features different from those detected at diagnosis. The evolving focus is on single-cell analytical tools to fully capture the pathobiological heterogeneity of AML. We leveraged CyTOF to interrogate the proteomic heterogeneity in patients with R/R AML, which could potentially permit the design of rational combinatorial therapeutic approaches targeting vulnerabilities in these cells.. Methods: To dissect AML heterogeneity and its contribution to treatment failure, we designed a 51-parameter CyTOF panel and interrogated cellular hierarchy, major immune phenotypes, anti-apoptotic molecules, signaling pathways, exhaustion markers and attractive targets for CAR T-cell therapy in R/R AML (n:13). Results: First, we generated two-dimensional t-SNE maps and observed that the leukemic bone marrow compartment harbored immature (CD34+CD38- and CD34+CD38+) and mature leukemic blasts (CD33+CD34-) and major immune subsets. Constitutively active signaling pathways characterized by high levels of p-4EBP1, p-MEK1/2, p-S6 and p-AKT, marked immature and mature leukemia cells and comparative analysis revealed that monocytic blasts harbored more active signaling networks. The proportions of these subpopulations varied significantly across patients. We initially assessed the distribution of anti-apoptotic molecules across these leukemia compartments. Strikingly, Bcl-2 levels were considerably high within less-differentiated leukemic cell compartments and CD68 expressing leukemic blasts with monocytic differentiation had significantly lower levels of Bcl-2. This suggests that differentiated leukemic cells could preferentially survive under selection pressure of Bcl-2 inhibitors. On the contrary, we observed a trend towards higher Mcl-1 levels in differentiated leukemia cells. These findings provide a rationale for combining therapeutic modalities to target different leukemia subpopulations. Indeed, Bcl-2 and Mcl-1 inhibitors (Venetoclax and AZD5991) resulted in highly synergistic effects in AML PDX models. Hence, this analysis supports the hypothesis that Mcl-1 overexpression is a resistance factor to Bcl-2 inhibition, usually understood as developing in the same cell. Next, we assessed expression patterns of putative CAR T-cell targets expressed on leukemic cells and identified significant variegated expression patterns in R/R AML samples. CD123 demonstrated patchy distribution across immature and differentiated leukemic blasts while CD33 expression was the main characteristic of differentiated leukemic blasts. Of note, the immature leukemia compartment demonstrated variable levels of CD33 and complete lack of CD33 on CD34+ leukemic cells was observed in a subgroup of patients. CLL-1 was uniformly expressed across all leukemia compartments, but was not ubiquitously expressed in all patient samples, revealing substantial interpatient heterogeneity in R/R AML and highlighting the concept of targeting at least two antigens concomitantly. Lastly, we sought to undertake high-dimensional assessment of immune compartments to identify major immune phenotypes in heavily treated R/R AML patients and discover the link between immune phenotypes and AML-associated traits. In line with leukemia cell heterogeneity, we found a significant degree of variation in immune cell composition among patients. Inhibitory molecules PD-1 and TIGIT were significantly expressed on CD4 and CD8 T-cells respectively, providing a rationale for use of combinatorial immunotherapeutic approach for the treatment of AML. Conclusion: Single-cell profiling of R/R AML using CyTOF reveals significantly heterogeneous expression patterns of molecules targeted by BH3 mimetics (Bcl-2 and Mcl-1), CAR T-cells, and other antibody-based immunotherapeutic therapies. This approach provides a rationale to develop combinatorial therapeutic approaches targeting distinct leukemia sub-populations with discrete expression patterns of established and novel putative targets. An example is the combined targeting of Bcl-2 and Mcl-1, which are differentially expressed in early and more differentiated leukemia subpopulations. Disclosures Carter: AstraZeneca: Research Funding; Syndax: Research Funding; Amgen: Research Funding; Ascentage: Research Funding. Andreeff:Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Amgen: Research Funding.

AML-354: High-Dimensional Single-Cell Proteomic Analysis Reveals Therapeutic Vulnerabilities in Patients with Relapsed/Refractory AML

We designed a 51-parameter mass cytometry panel and interrogated acute myeloid leukemia (AML) heterogeneity to assess cellular hierarchy, anti-apoptotic molecules, exhaustion markers and targets for CAR T-cell therapy in R/R AML. We generated two-dimensional t-SNE maps to visualize distinct populations and observed significant heterogeneity among patients with regards to leukemic compartment composition. Notably, the leukemic bone marrow compartment harbored leukemia stem cells (CD34+CD38-), immature leukemic cells (CD34+CD38+), more differentiated leukemic blasts (CD33+CD34-) and major immune subsets. We initially assessed the distribution of anti-apoptotic molecules across leukemia compartments. Strikingly, Bcl-2 levels were considerably high within less-differentiated leukemic cell compartments and leukemic blasts with monocytic differentiation had significantly low levels of Bcl-2. On the contrary, we observed a trend towards higher Mcl-1 levels in differentiated leukemic cells. These findings provide a rationale for combining therapeutic modalities to target different leukemia subpopulations. Indeed, Bcl-2 and Mcl-1 inhibitors resulted in highly synergistic effects in AML PDX models (Carter et.al 2018, 2019). Of interest, Bcl-xl was ubiquitously expressed across leukemic and immune cell clusters. Next, we assessed expression patterns of CAR T-cell targets expressed on leukemic cells and identified significant variegated expression patterns for all CAR T-cell therapy targets in R/R AML samples. CD123 demonstrated patchy distribution across immature and differentiated leukemic blasts while CD33 expression was the main characteristic of differentiated leukemic blasts. Of note, the immature leukemia compartment demonstrated variable levels of CD33 and complete lack of CD33 on CD34+ leukemic cells was observed in a subgroup of patients. CLL-1 expression was the most promising marker and expressed across almost all leukemia compartments. Nevertheless, CLL-1 expression on leukemic cells was not ubiquitous in a small group of patients. In line with leukemic cell heterogeneity, we found a significant degree of variation in immune cell composition. Inhibitory molecules PD-1 and TIGIT were significantly expressed on CD4 and CD8 T-cells respectively. Single-cell profiling of R/R AML using mass cytometry reveals significant heterogeneous expression patterns of molecules targeted by CAR T-cells, immunotherapeutic approaches and targeted therapies. This approach provides a rationale to adopt combinatorial therapeutic approaches to target distinct leukemic populations with widely varying target expression.

Histone chaperones and the Rrm3p helicase regulate flocculation in S. cerevisiae

BackgroundBiofilm formation or flocculation is a major phenotype in wild type budding yeasts but rarely seen in laboratory yeast strains. Here, we analysed flocculation phenotypes and the expression of FLO genes in laboratory strains with various genetic backgrounds.ResultsWe show that mutations in histone chaperones, the helicase RRM3 and the Histone Deacetylase HDA1 de-repress the FLO genes and partially reconstitute flocculation. We demonstrate that the loss of repression correlates to elevated expression of several FLO genes, to increased acetylation of histones at the promoter of FLO1 and to variegated expression of FLO11. We show that these effects are related to the activity of CAF-1 at the replication forks. We also demonstrate that nitrogen starvation or inhibition of histone deacetylases do not produce flocculation in W303 and BY4742 strains but do so in strains compromised for chromatin maintenance. Finally, we correlate the de-repression of FLO genes to the loss of silencing at the subtelomeric and mating type gene loci.ConclusionsWe conclude that the deregulation of chromatin maintenance and transmission is sufficient to reconstitute flocculation in laboratory yeast strains. Consequently, we propose that a gain in epigenetic silencing is a major contributing factor for the loss of flocculation phenotypes in these strains. We suggest that flocculation in yeasts provides an excellent model for addressing the challenging issue of how epigenetic mechanisms contribute to evolution.

A Heterochromatin Domain Forms Gradually at a New Telomere and Is Dynamic at Stable Telomeres.

Heterochromatin domains play important roles in chromosome biology, organismal development, and aging, including centromere function, mammalian female X chromosome inactivation, and senescence-associated heterochromatin foci. In the fission yeast Schizosaccharomyces pombe and metazoans, heterochromatin contains histone H3 that is dimethylated at lysine 9. While factors required for heterochromatin have been identified, the dynamics of heterochromatin formation are poorly understood. Telomeres convert adjacent chromatin into heterochromatin. To form a new heterochromatic region in S. pombe, an inducible DNA double-strand break (DSB) was engineered next to 48 bp of telomere repeats in euchromatin, which caused formation of a new telomere and the establishment and gradual spreading of a new heterochromatin domain. However, spreading was dynamic even after the telomere had reached its stable length, with reporter genes within the heterochromatin domain showing variegated expression. The system also revealed the presence of repeats located near the boundaries of euchromatin and heterochromatin that are oriented to allow the efficient healing of a euchromatic DSB to cap the chromosome end with a new telomere. Telomere formation in S. pombe therefore reveals novel aspects of heterochromatin dynamics and fail-safe mechanisms to repair subtelomeric breaks, with implications for similar processes in metazoan genomes.

Abstract IA22: Targeting CARs to the TRAC locus enhances T-cell potency

Abstract Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumor rejection. The most successful CARs used to date are those targeting CD19, which offer the prospect of complete remissions in patients with chemorefractory/relapsed B cell malignancies. CARs are typically transduced into patient T cells using γ-retroviral or other randomly-integrating retroviral vectors (RVs), which may result in variegated CAR expression and transcriptional silencing. Recent advances in genome editing enable efficient sequence-specific modifications in human primary cells, including site-specific transgene integration into the CCR5 and AAVS1 loci in T lymphocytes. Using CRISPR/Cas9 to edit human T cells, we have established conditions yielding efficient target gene disruption and CAR insertion in a single step. We have found that directing a CD19 CAR to the human T-cell receptor (TCR) alpha chain (TRAC) locus not only results in efficient and uniform CAR expression in human peripheral blood T cells, but, remarkably, also enhances T-cell potency, vastly outperforming that of conventionally generated CAR T cells. We further show that CAR gene expression under the control of the TCR alpha promoter minimizes tonic signaling and allows effective regulation of CAR expression. These findings provide important insights into CAR immunobiology and highlight the potential of CRISPR/Cas9 genome editing to advance immunotherapies. Citation Format: Michel Sadelain, Justin Eyquem. Targeting CARs to the TRAC locus enhances T-cell potency [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA22.

An ApiAP2 member regulates expression of clonally variant genes of the human malaria parasite Plasmodium falciparum

Variegated surface antigen expression is key to chronic infection and pathogenesis of the human malaria parasite Plasmodium falciparum. This protozoan parasite expresses distinct surface molecules that are encoded by clonally variant gene families such as var, rif and stevor. The molecular mechanisms governing activation of individual members remain ill-defined. To investigate the molecular events of the initial transcriptional activation process we focused on a member of the apicomplexan ApiAP2 transcription factor family predicted to bind to the 5′ upstream regions of the var gene family, AP2-exp (PF3D7_1466400). Viable AP2-exp mutant parasites rely on expressing no less than a short truncated protein including the N-terminal AP2 DNA-binding domain. RNA-seq analysis in mutant parasites revealed transcriptional changes in a subset of exported proteins encoded by clonally variant gene families. Upregulation of RIFINs and STEVORs was validated at the protein levels. In addition, morphological alterations were observed on the surface of the host cells infected by the mutants. This work points to a complex regulatory network of clonally variant gene families in which transcription of a subset of members is regulated by the same transcription factor. In addition, we highlight the importance of the non-DNA binding AP2 domain in functional gene regulation.

Variegated gene expression and Sox-13 mediated regulation of WC1, a hybrid PRR/Co-receptor exclusive to memory gd T cells

Abstract γδ T cells have broader reactivity and respond more rapidly compared to αβ T cells which are restricted to peptide bound to MHC. Some γδ T cells bear lineage-specific glycoproteins known as WC1 that are coded by a multigenic family composed of 13 members. Structurally, WC1 molecules have 6–11 SRCR domains, followed by a conserved transmembrane and a variable cytoplasmic tail. γδ T cells expressing these molecules can be further divided into subsets that express either WC1-3 or WC1-4, a dichotomy analogous to CD4 and CD8 molecules and similarly set up in the thymus. While WC1-3+ and WC1-4+ γδ T cells share a restricted repertoire of the γδ TCR genes, expression of WC1-3 vs. WC1-4 determines whether the γδ T cell is activated by Leptospira; reactivity correlates with expression of WC1-3 and the ability of its SRCR domains to bind to Leptospira. ShRNA-mediated knockdown of WC1-3 significantly reduces the ability of WC1-3 expressing γδ T cells to respond to the bacteria, guiding us to a hypothesis that WC1 is essential for recognition of bacterial pathogens by γδ T cells and that the WC1 family members expressed by a particular cell will determine its ability to respond to an infection. Evaluation of WC1 gene expression among individual γδ T cells indicates that WC1-3 and WC1-4 are always expressed reciprocally, establishing functional subsets. These γδ T cell subsets appear to be set in the thymus, alluding to the transcription factor occupancy of WC1-3 vs WC1-4. Occupancy of candidate Sox13 binding sites differ between WC1-3 and WC1-4 loci, suggesting that Sox13 may play an important role in determining the ability of mature γδ T cells to respond to bacteria.

Natural Killer Cell Diversity in Viral Infection: Why and How Much?

Natural killer cells are a diverse group of innate lymphocytes that are specialized to rapidly respond to cancerous or virus-infected cells. NK cell function is controlled by the integration of signals from activating and inhibitory receptors expressed at the cell surface. Variegated expression patterns of these activating and inhibitory receptors at the single cell level leads to a highly diverse NK cell repertoire. Here I review the factors that influence NK cell repertoire diversity and its functional consequences for our ability to fight viruses.