Distinct kinesin motors drive two types of maize neocentromeres.

A maize chromosome variant called abnormal chromosome 10 (Ab10) converts knobs on chromosome arms into neocentromeres, causing their preferential segregation to egg cells in a process known as meiotic drive. We previously demonstrated that the gene Kinesin driver (Kindr) on Ab10 encodes a kinesin-14 required to mobilize neocentromeres made up of the major tandem repeat knob180. Here we describe a second kinesin-14 gene, TR-1 kinesin (Trkin), that is required to mobilize neocentromeres made up of the minor tandem repeat TR-1. Trkin lies in a 4-Mb region of Ab10 that is not syntenic with any other region of the maize genome and shows extraordinary sequence divergence from Kindr and other kinesins in plants. Despite its unusual structure, Trkin encodes a functional minus end-directed kinesin that specifically colocalizes with TR-1 in meiosis, forming long drawn out neocentromeres. TRKIN contains a nuclear localization signal and localizes to knobs earlier in prophase than KINDR. The fact that TR-1 repeats often co-occur with knob180 repeats suggests that the current role of the TRKIN/TR-1 system is to facilitate the meiotic drive of the KINDR/knob180 system.

Radiotherapy Eradicates Malignant T Cells and Is Associated with Improved Survival in Early-Stage Mycosis Fungoides.

PURPOSE: Mycosis fungoides is the most common subtype of cutaneous T-cell lymphoma. Skin-directed treatments often improve but do not cure mycosis fungoides skin lesions. The purpose of this study was to (i) assess whether remission was associated with malignant T-cell clone depletion at treated sites using either low-dose radiotherapy (LDRT, 8 Gy) or topical steroids and (ii) assess whether a clone-ablative therapy, like LDRT, is associated with overall survival in patients with high-risk early-stage CTCL. EXPERIMENTAL DESIGN: Pre- and posttreatment biopsies from 20 lesional skin samples of 18 patients with mycosis fungoides who received either 8 Gy LDRT (n = 16) or topical steroids (n = 4) underwent high-throughput T-cell receptor sequencing of the TCRB gene to quantify the malignant T-cell clone. For the retrospective chart review, overall survival of 47 high-risk early-stage patients was compared between patients who did or did not receive radiation. RESULTS: LDRT eradicated the clone in 5 of 16 lesions and reduced it >90% in 11 of 16; there were no recurrences in these lesions. Patients treated with topical steroids appeared to clinically improve, but the malignant clone persisted. We found that the number of residual malignant T cells predicted lesion recurrence. A retrospective review showed that early-stage high-risk patients who received radiation as part of their treatment regimen had prolonged overall survival compared with patients who did not. CONCLUSIONS: These findings demonstrate that LDRT can eradicate malignant T cells in mycosis fungoides, provides robust disease control, and is associated with improved survival in high-risk early-stage patients.

Benign T cells drive clinical skin inflammation in cutaneous T cell lymphoma.

Psoralen plus UVA (PUVA) is an effective therapy for mycosis fungoides (MF), the skin-limited variant of cutaneous T cell lymphoma (CTCL). In low-burden patients, PUVA reduced or eradicated malignant T cells and induced clonal expansion of CD8+ T cells associated with malignant T cell depletion. High-burden patients appeared to clinically improve but large numbers of malignant T cells persisted in skin. Clinical improvement was linked to turnover of benign T cell clones but not to malignant T cell reduction. Benign T cells were associated with the Th2-recruiting chemokine CCL18 before therapy and with the Th1-recruiting chemokines CXCL9, CXCL10, and CXCL11 after therapy, suggesting a switch from Th2 to Th1. Inflammation was correlated with OX40L and CD40L gene expression; immunostaining localized these receptors to CCL18-expressing c-Kit+ dendritic cells that clustered together with CD40+OX40+ benign and CD40+CD40L+ malignant T cells, creating a proinflammatory synapse in skin. Our data suggest that visible inflammation in CTCL results from the recruitment and activation of benign T cells by c-Kit+OX40L+CD40L+ dendritic cells and that this activation may provide tumorigenic signals. Targeting c-Kit, OX40, and CD40 signaling may be novel therapeutic avenues for the treatment of MF.

High-throughput sequencing of the T cell receptor ß gene identifies aggressive early-stage mycosis fungoides.

Mycosis fungoides (MF), the most common cutaneous T cell lymphoma (CTCL) is a malignancy of skin-tropic memory T cells. Most MF cases present as early stage (stage I A/B, limited to the skin), and these patients typically have a chronic, indolent clinical course. However, a small subset of early-stage cases develop progressive and fatal disease. Because outcomes can be so different, early identification of this high-risk population is an urgent unmet clinical need. We evaluated the use of next-generation high-throughput DNA sequencing of the T cell receptor ß gene (TCRB) in lesional skin biopsies to predict progression and survival in a discovery cohort of 208 patients with CTCL (177 with MF) from a 15-year longitudinal observational clinical study. We compared these data to the results in an independent validation cohort of 101 CTCL patients (87 with MF). The tumor clone frequency (TCF) in lesional skin, measured by high-throughput sequencing of the TCRB gene, was an independent prognostic factor of both progression-free and overall survival in patients with CTCL and MF in particular. In early-stage patients, a TCF of >25% in the skin was a stronger predictor of progression than any other established prognostic factor (stage IB versus IA, presence of plaques, high blood lactate dehydrogenase concentration, large-cell transformation, or age). The TCF therefore may accurately predict disease progression in early-stage MF. Early identification of patients at high risk for progression could help identify candidates who may benefit from allogeneic hematopoietic stem cell transplantation before their disease becomes treatment-refractory.

A Kinesin-14 Motor Activates Neocentromeres to Promote Meiotic Drive in Maize.

Maize abnormal chromosome 10 (Ab10) encodes a classic example of true meiotic drive that converts heterochromatic regions called knobs into motile neocentromeres that are preferentially transmitted to egg cells. Here, we identify a cluster of eight genes on Ab10, called the Kinesin driver (Kindr) complex, that are required for both neocentromere motility and preferential transmission. Two meiotic drive mutants that lack neocentromere activity proved to be kindr epimutants with increased DNA methylation across the entire gene cluster. RNAi of Kindr induced a third epimutant and corresponding loss of meiotic drive. Kinesin gliding assays and immunolocalization revealed that KINDR is a functional minus-end-directed kinesin that localizes specifically to knobs containing 180 bp repeats. Sequence comparisons suggest that Kindr diverged from a Kinesin-14A ancestor ∼12 mya and has driven the accumulation of > 500 Mb of knob repeats and affected the segregation of thousands of genes linked to knobs on all 10 chromosomes.

Fitness Costs and Variation in Transmission Distortion Associated with the Abnormal Chromosome 10 Meiotic Drive System in Maize.

Meiotic drive describes a process whereby selfish genetic elements are transmitted at levels greater than Mendelian expectations. Maize abnormal chromosome 10 (Ab10) encodes a meiotic drive system that exhibits strong preferential segregation through female gametes. We performed transmission assays on nine Ab10 chromosomes from landraces and teosinte lines and found a transmission advantage of 62-79% in heterozygotes. Despite this transmission advantage, Ab10 is present at low frequencies in natural populations, suggesting that it carries large negative fitness consequences. We measured pollen transmission, the percentage of live pollen, seed production, and seed size to estimate several of the possible fitness effects of Ab10. We found no evidence that Ab10 affects pollen transmission, i.e., Ab10 and N10 pollen are transmitted equally from heterozygous fathers. However, at the diploid (sporophyte) level, both heterozygous and homozygous Ab10-I-MMR individuals show decreased pollen viability, decreased seed set, and decreased seed weight. The observed fitness costs can nearly but not entirely account for the observed frequencies of Ab10. Sequence analysis shows a surprising amount of molecular variation among Ab10 haplotypes, suggesting that there may be other phenotypic variables that contribute to the low but stable equilibrium frequencies.

Clinically resolved psoriatic lesions contain psoriasis-specific IL-17-producing αß T cell clones.

In psoriasis, an IL-17-mediated inflammatory skin disease, skin lesions resolve with therapy, but often recur in the same locations when therapy is discontinued. We propose that residual T cell populations in resolved psoriatic lesions represent the pathogenic T cells of origin in this disease. Utilizing high-throughput screening (HTS) of the T cell receptor (TCR) and immunostaining, we found that clinically resolved psoriatic lesions contained oligoclonal populations of T cells that produced IL-17A in both resolved and active psoriatic lesions. Putative pathogenic clones preferentially utilized particular Vß and Vα subfamilies. We identified 15 TCRß and 4 TCRα antigen receptor sequences shared between psoriasis patients and not observed in healthy controls or other inflammatory skin conditions. To address the relative roles of αß versus γδ T cells in psoriasis, we carried out TCR/δ HTS. These studies demonstrated that the majority of T cells in psoriasis and healthy skin are αß T cells. γδ T cells made up 1% of T cells in active psoriasis, less than 1% in resolved psoriatic lesions, and less than 2% in healthy skin. All of the 70 most frequent putative pathogenic T cell clones were αß T cells. In summary, IL-17-producing αß T cell clones with psoriasis-specific antigen receptors exist in clinically resolved psoriatic skin lesions. These cells likely represent the disease-initiating pathogenic T cells in psoriasis, suggesting that lasting control of this disease will require suppression of these resident T cell populations.

RFWD3-Dependent Ubiquitination of RPA Regulates Repair at Stalled Replication Forks.

We have used quantitative proteomics to profile ubiquitination in the DNA damage response (DDR). We demonstrate that RPA, which functions as a protein scaffold in the replication stress response, is multiply ubiquitinated upon replication fork stalling. Ubiquitination of RPA occurs on chromatin, involves sites outside its DNA binding channel, does not cause proteasomal degradation, and increases under conditions of fork collapse, suggesting a role in repair at stalled forks. We demonstrate that the E3 ligase RFWD3 mediates RPA ubiquitination. RFWD3 is necessary for replication fork restart, normal repair kinetics during replication stress, and homologous recombination (HR) at stalled replication forks. Mutational analysis suggests that multisite ubiquitination of the entire RPA complex is responsible for repair at stalled forks. Multisite protein group sumoylation is known to promote HR in yeast. Our findings reveal a similar requirement for multisite protein group ubiquitination during HR at stalled forks in mammalian cells.

TCR sequencing facilitates diagnosis and identifies mature T cells as the cell of origin in CTCL.

Early diagnosis of cutaneous T cell lymphoma (CTCL) is difficult and takes on average 6 years after presentation, in part because the clinical appearance and histopathology of CTCL can resemble that of benign inflammatory skin diseases. Detection of a malignant T cell clone is critical in making the diagnosis of CTCL, but the T cell receptor γ (TCRγ) polymerase chain reaction (PCR) analysis in current clinical use detects clones in only a subset of patients. High-throughput TCR sequencing (HTS) detected T cell clones in 46 of 46 CTCL patients, was more sensitive and specific than TCRγ PCR, and successfully discriminated CTCL from benign inflammatory diseases. HTS also accurately assessed responses to therapy and facilitated diagnosis of disease recurrence. In patients with new skin lesions and no involvement of blood by flow cytometry, HTS demonstrated hematogenous spread of small numbers of malignant T cells. Analysis of CTCL TCRγ genes demonstrated that CTCL is a malignancy derived from mature T cells. There was a maximal T cell density in skin in benign inflammatory diseases that was exceeded in CTCL, suggesting that a niche of finite size may exist for benign T cells in skin. Last, immunostaining demonstrated that the malignant T cell clones in mycosis fungoides and leukemic CTCL localized to different anatomic compartments in the skin. In summary, HTS accurately diagnosed CTCL in all stages, discriminated CTCL from benign inflammatory skin diseases, and provided insights into the cell of origin and location of malignant CTCL cells in skin.

Epstein-Barr virus nuclear antigen leader protein localizes to promoters and enhancers with cell transcription factors and EBNA2.

Epstein-Barr virus (EBV) nuclear antigens EBNALP (LP) and EBNA2 (E2) are coexpressed in EBV-infected B lymphocytes and are critical for lymphoblastoid cell line outgrowth. LP removes NCOR and RBPJ repressive complexes from promoters, enhancers, and matrix-associated deacetylase bodies, whereas E2 activates transcription from distal enhancers. LP ChIP-seq analyses identified 19,224 LP sites of which ~50% were ± 2 kb of a transcriptional start site. LP sites were enriched for B-cell transcription factors (TFs), YY1, SP1, PAX5, BATF, IRF4, ETS1, RAD21, PU.1, CTCF, RBPJ, ZNF143, SMC3, NFκB, TBLR, and EBF. E2 sites were also highly enriched for LP-associated cell TFs and were more highly occupied by RBPJ and EBF. LP sites were highly marked by H3K4me3, H3K27ac, H2Az, H3K9ac, RNAPII, and P300, indicative of activated transcription. LP sites were 29% colocalized with E2 (LP/E2). LP/E2 sites were more similar to LP than to E2 sites in associated cell TFs, RNAPII, P300, and histone H3K4me3, H3K9ac, H3K27ac, and H2Az occupancy, and were more highly transcribed than LP or E2 sites. Gene affected by CTCF and LP cooccupancy were more highly expressed than genes affected by CTCF alone. LP was at myc enhancers and promoters and of MYC regulated ccnd2, 23 med complex components, and MYC regulated cell survival genes, igf2r and bcl2. These data implicate LP and associated TFs and DNA looping factors CTCF, RAD21, SMC3, and YY1/INO80 chromatin-remodeling complexes in repressor depletion and gene activation necessary for lymphoblastoid cell line growth and survival.

Characterization of NMDAR-Independent Learning in the Hippocampus.

It is currently thought that memory formation requires the activation of NMDA receptors (NMDARs) in the hippocampus. However, recent studies indicate that these receptors are not necessary for all forms of learning. The current experiments examine this issue using context fear conditioning in mice. First, we show that context fear can be acquired without NMDAR activation in previously trained animals. Mice trained in one environment (context A) are subsequently able to learn about a second environment (context B) in the presence of NMDAR antagonists. Second, we demonstrate that NMDAR-independent learning requires the hippocampus and is dependent on protein synthesis. However, unlike NMDAR-dependent learning, it is not contingent on the expression of activity-regulated cytoskeleton-associated protein (Arc). Lastly, we present data that suggests NMDAR-independent learning is only observed when recently stimulated neurons are reactivated during conditioning. These data suggest that context fear conditioning modifies synaptic plasticity mechanisms in the hippocampus and allows subsequent learning to occur in the absence of NMDAR activation.