Dual and opposing roles for the kinesin-2 motor, KIF17, in Hedgehog-dependent cerebellar development.

While the kinesin-2 motors KIF3A and KIF3B have essential roles in ciliogenesis and Hedgehog (HH) signal transduction, potential role(s) for another kinesin-2 motor, KIF17, in HH signaling have yet to be explored. Here, we investigated the contribution of KIF17 to HH-dependent cerebellar development, where Kif17 is expressed in both HH-producing Purkinje cells and HH-responding cerebellar granule neuron progenitors (CGNPs). Germline Kif17 deletion in mice results in cerebellar hypoplasia due to reduced CGNP proliferation, a consequence of decreased HH pathway activity mediated through decreased Sonic HH (SHH) protein. Notably, Purkinje cell-specific Kif17 deletion partially phenocopies Kif17 germline mutants. Unexpectedly, CGNP-specific Kif17 deletion results in the opposite phenotype-increased CGNP proliferation and HH target gene expression due to altered GLI transcription factor processing. Together, these data identify KIF17 as a key regulator of HH-dependent cerebellar development, with dual and opposing roles in HH-producing Purkinje cells and HH-responding CGNPs.

SPR-1/CoREST facilitates the maternal epigenetic reprogramming of the histone demethylase SPR-5/LSD1.

Maternal reprogramming of histone methylation is critical for reestablishing totipotency in the zygote, but how histone-modifying enzymes are regulated during maternal reprogramming is not well characterized. To address this gap, we asked whether maternal reprogramming by the H3K4me1/2 demethylase SPR-5/LSD1/KDM1A, is regulated by the chromatin co-repressor protein, SPR-1/CoREST, in Caenorhabditis elegans and mice. In C. elegans, SPR-5 functions as part of a reprogramming switch together with the H3K9 methyltransferase MET-2. By examining germline development, fertility, and gene expression in double mutants between spr-1 and met-2, as well as fertility in double mutants between spr-1 and spr-5, we find that loss of SPR-1 results in a partial loss of SPR-5 maternal reprogramming function. In mice, we generated a separation of function Lsd1 M448V point mutation that compromises CoREST binding, but only slightly affects LSD1 demethylase activity. When maternal LSD1 in the oocyte is derived exclusively from this allele, the progeny phenocopy the increased perinatal lethality that we previously observed when LSD1 was reduced maternally. Together, these data are consistent with CoREST having a conserved function in facilitating maternal LSD1 epigenetic reprogramming.

Landscape and selection of vaccine epitopes in SARS-CoV-2.

BACKGROUND: Early in the pandemic, we designed a SARS-CoV-2 peptide vaccine containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation. The rationale for this design was to drive both humoral and cellular immunity with high specificity while avoiding undesired effects such as antibody-dependent enhancement (ADE). METHODS: We explored the set of computationally predicted SARS-CoV-2 HLA-I and HLA-II ligands, examining protein source, concurrent human/murine coverage, and population coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, sequence conservation, source protein abundance, and coverage of high frequency HLA alleles. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering for surface accessibility, sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites. RESULTS: From 58 initial candidates, three B cell epitope regions were identified. From 3730 (MHC-I) and 5045 (MHC-II) candidate ligands, 292 CD8+ and 284 CD4+ T cell epitopes were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we proposed a set of 22 SARS-CoV-2 vaccine peptides for use in subsequent murine studies. We curated a dataset of ~ 1000 observed T cell epitopes from convalescent COVID-19 patients across eight studies, showing 8/15 recurrent epitope regions to overlap with at least one of our candidate peptides. Of the 22 candidate vaccine peptides, 16 (n = 10 T cell epitope optimized; n = 6 B cell epitope optimized) were manually selected to decrease their degree of sequence overlap and then synthesized. The immunogenicity of the synthesized vaccine peptides was validated using ELISpot and ELISA following murine vaccination. Strong T cell responses were observed in 7/10 T cell epitope optimized peptides following vaccination. Humoral responses were deficient, likely due to the unrestricted conformational space inhabited by linear vaccine peptides. CONCLUSIONS: Overall, we find our selection process and vaccine formulation to be appropriate for identifying T cell epitopes and eliciting T cell responses against those epitopes. Further studies are needed to optimize prediction and induction of B cell responses, as well as study the protective capacity of predicted T and B cell epitopes.

Caenorhabditis elegans establishes germline versus soma by balancing inherited histone methylation.

Formation of a zygote is coupled with extensive epigenetic reprogramming to enable appropriate inheritance of histone methylation and prevent developmental delays. In Caenorhabditis elegans, this reprogramming is mediated by the H3K4me2 demethylase SPR-5 and the H3K9 methyltransferase, MET-2. In contrast, the H3K36 methyltransferase MES-4 maintains H3K36me2/3 at germline genes between generations to facilitate re-establishment of the germline. To determine whether the MES-4 germline inheritance pathway antagonizes spr-5; met-2 reprogramming, we examined the interaction between these two pathways. We found that the developmental delay of spr-5; met-2 mutant progeny is associated with ectopic H3K36me3 and the ectopic expression of MES-4-targeted germline genes in somatic tissues. Furthermore, the developmental delay is dependent upon MES-4 and the H3K4 methyltransferase, SET-2. We propose that MES-4 prevents crucial germline genes from being repressed by antagonizing maternal spr-5; met-2 reprogramming. Thus, the balance of inherited histone modifications is necessary to distinguish germline versus soma and prevent developmental delay.This article has an associated 'The people behind the papers' interview.

Repressive H3K9me2 protects lifespan against the transgenerational burden of COMPASS activity in C. elegans.

In Caenorhabditis elegans, mutations in WDR-5 and other components of the COMPASS H3K4 methyltransferase complex extend lifespan and enable its inheritance. Here, we show that wdr-5 mutant longevity is itself a transgenerational trait that corresponds with a global enrichment of the heterochromatin factor H3K9me2 over twenty generations. In addition, we find that the transgenerational aspects of wdr-5 mutant longevity require the H3K9me2 methyltransferase MET-2, and can be recapitulated by removal of the putative H3K9me2 demethylase JHDM-1. Finally, we show that the transgenerational acquisition of longevity in jhdm-1 mutants is associated with accumulating genomic H3K9me2 that is inherited by their long-lived wild-type descendants at a subset of loci. These results suggest that heterochromatin facilitates the transgenerational establishment and inheritance of a complex trait. Based on these results, we propose that transcription-coupled H3K4me via COMPASS limits lifespan by encroaching upon domains of heterochromatin in the genome.

The Pipeline CURE: An Iterative Approach to Introduce All Students to Research Throughout a Biology Curriculum.

Participation in research provides personal and professional benefits for undergraduates. However, some students face institutional barriers that prevent their entry into research, particularly those from underrepresented groups who may stand to gain the most from research experiences. Course-based undergraduate research experiences (CUREs) effectively scale research availability, but many only last for a single semester, which is rarely enough time for a novice to develop proficiency. To address these challenges, we present the Pipeline CURE, a framework that integrates a single research question throughout a biology curriculum. Students are introduced to the research system - in this implementation, C. elegans epigenetics research - with their first course in the major. After revisiting the research system in several subsequent courses, students can choose to participate in an upper-level research experience. In the Pipeline, students build resilience via repeated exposure to the same research system. Its iterative, curriculum-embedded approach is flexible enough to be implemented at a range of institutions using a variety of research questions. By uniting evidence-based teaching methods with ongoing scientific research, the Pipeline CURE provides a new model for overcoming barriers to participation in undergraduate research.

An ancient yet flexible cis-regulatory architecture allows localized Hedgehog tuning by patched/Ptch1.

The Hedgehog signaling pathway is part of the ancient developmental-evolutionary animal toolkit. Frequently co-opted to pattern new structures, the pathway is conserved among eumetazoans yet flexible and pleiotropic in its effects. The Hedgehog receptor, Patched, is transcriptionally activated by Hedgehog, providing essential negative feedback in all tissues. Our locus-wide dissections of the cis-regulatory landscapes of fly patched and mouse Ptch1 reveal abundant, diverse enhancers with stage- and tissue-specific expression patterns. The seemingly simple, constitutive Hedgehog response of patched/Ptch1 is driven by a complex regulatory architecture, with batteries of context-specific enhancers engaged in promoter-specific interactions to tune signaling individually in each tissue, without disturbing patterning elsewhere. This structure-one of the oldest cis-regulatory features discovered in animal genomes-explains how patched/Ptch1 can drive dramatic adaptations in animal morphology while maintaining its essential core function. It may also suggest a general model for the evolutionary flexibility of conserved regulators and pathways.

The heterotrimeric kinesin-2 complex interacts with and regulates GLI protein function.

GLI transport to the primary cilium and nucleus is required for proper Hedgehog (HH) signaling; however, the mechanisms that mediate these trafficking events are poorly understood. Kinesin-2 motor proteins regulate ciliary transport of cargo, yet their role in GLI protein function remains unexplored. To examine a role for the heterotrimeric KIF3A-KIF3B-KAP3 kinesin-2 motor complex in regulating GLI activity, we performed a series of structure-function analyses using biochemical, cell signaling and in vivo approaches that define novel specific interactions between GLI proteins and two components of this complex, KAP3 and KIF3A. We find that all three mammalian GLI proteins interact with KAP3 and we map specific interaction sites in both proteins. Furthermore, we find that GLI proteins interact selectively with KIF3A, but not KIF3B, and that GLI interacts synergistically with KAP3 and KIF3A. Using a combination of cell signaling assays and chicken in ovo electroporation, we demonstrate that KAP3 interactions restrict GLI activator function but not GLI repressor function. These data suggest that GLI interactions with KIF3A-KIF3B-KAP3 complexes are essential for proper GLI transcriptional activity.