Epigenetics & Chromatin

Background: The spatial organization of the human genome emerges from an enzyme-guided self-assembly process in which chromatin has a bidirectional relationship with transcription. Central to this architecture are chromatin packing domains, 50-200 nm structures exhibiting mass-scaling behavior. Nascent domains arise through transcription and cohesin/CTCF-mediated loop extrusion, while their maturation depends on conditions that permit differential access of epigenetic enzymes to their dense cores and continuously lower density periphery, resulting in the accumulation of heterochromatic marks in domain cores and euchromatic marks outside of the domain cores. In turn, packing domains support transcription by creating an "ideal zone" peripherally to their cores due to a nonmonotonic dependence of transcription rate on local chromatin density. Results: We investigated the role of heterochromatin protein 1 alpha (HP1), a key chromatin crosslinking factor with affinity to H3K9me3 marks, in maintaining packing domain organization. Paradoxically, short-term HP1 degradation resulted in overall downregulation of genes, contrasting with the canonical view that repression of heterochromatin allows chromatin to become more accessible for transcription. While some packing domains persisted, heterochromatic cores of dense packing domains were disrupted. When exposed to hypoxia, HP1-depleted cells exhibited a dampened transcriptional response, showcasing that when domain maturation is disrupted, stress response pathways are not effectively activated. Conclusions: This work highlights HP1 crosslinking as an essential component of domain maturation, as HP1 loss disrupts domain geometry and inhibits efficient transcriptional reprogramming. Our findings suggest an intriguing perspective on the potential relationship between chromatin architecture and gene regulation that could inform chromatin engineering-based treatments.

Chromatin profiling methods, such as ChIP-seq (chromatin immunoprecipitation followed by sequencing), are used to characterize the genomic localization of DNA-associated proteins. While ChIP and ChIP-seq have been used for decades, an orthogonal approach, CUT&Tag (Cleavage Under Targets and Tagmentation), is gaining popularity as an efficient and cost-effective alternative. Although previous comparative studies note discrepancies in signal-to-noise ratios and detection bias at certain genomic regions, many differences between ChIP-seq and CUT&Tag results remain largely unreconciled. Here, we systematically investigate the disagreeing signals captured by these two methods across well annotated genomic regions. We assess multiple histone mark profiles generated by different groups in two cell lines (K562 and MCF-7). Overall, our analysis indicates that compared to ChIP-seq, CUT&Tag may have limited sensitivity in low-GC environments and, as previously observed, increased signal in hyper-accessible chromatin. Notably, within GC-poor regions of active gene bodies and Polycomb-repressed domains, CUT&Tag exhibits a loss of H3K36me3 and H3K27me3 signal, respectively, where occupancy of these histone marks is otherwise expected. Further, active promoters with discrepant H3K4me3 and H3K27ac signal between the two assays differ systematically in GC content and chromatin accessibility. Promoters differentially enriched for CUT&Tag signal relative to ChIP-seq generally show higher, broader GC-content profiles and higher DNase-seq and ATAC-seq signal, while promoters enriched for ChIP-seq signal harbor the opposite characteristics. A local bias for high GC content and/or chromatin accessibility in CUT&Tag may also explain its differing signal patterns at nucleosome-depleted regions (NDRs) compared to ChIP-seq and MNase-seq promoter profiles. Altogether, our results highlight that studies focused on profiling the intensity and structure of histone modification occupancy can be sensitive to potential biases of CUT&Tag to GC content and chromatin accessibility. These characteristics should be accounted for when selecting a chromatin profiling approach, analyzing and interpreting data as well as drawing biological conclusions.

Developmental genes often acquire accessible chromatin prior to their activation, yet the mechanisms regulating this permissive state remain poorly understood. During spermatogenesis, many meiotic gene promoters display this poised state in spermatogonia, characterized by accessible promoters and paused RNA Pol II occupancy before their activation during meiosis. Here, we identify the NURF chromatin remodeling complex, comprised of SMARCA5 and its accessory subunit BPTF, as a key regulator of this process. We demonstrate that the transcription factor NFYA interacts with SMARCA5 and is required for its occupancy at poised meiotic gene promoters. Approximately one-third of poised meiotic gene promoters is co-occupied by NFYA, SMARCA5, and BPTF. Germline deletion of Smarca5 disrupts chromatin accessibility at poised meiotic promoters, impairs transcriptional program required for mitosis-to-meiosis transition, resulting in developmental arrest at meiotic entry. These findings identify NFYA-NURF as a key regulatory axis that regulates accessible chromatin at developmentally poised meiotic genes and primes them for activation later during meiosis.

The spatial organisation of eukaryotic genomes is critical for coordinating DNA-dependent processes such as transcription and replication. Cohesin contributes to this organisation by extruding chromatin loops; however, how cohesin achieves processive translocation along nucleosome-dense chromatin in vivo remains poorly understood. Here, we identify the histone chaperone FACT as a regulator of cohesin dynamics in human cells. We show that the FACT subunit SUPT16H colocalises and interacts with cohesin on chromatin. Depletion of FACT impairs cohesin translocation from its loading sites, leading to reduced chromatin looping and decreased boundary strength of topologically associating domains (TADs), as revealed by Hi-C. In addition, FACT depletion results in the accumulation of cohesin within gene bodies that are not engaged in loop formation, which is associated with reduced transcription. Importantly, degradation of cohesin in FACT-depleted cells restores transcriptional output, indicating that cohesin mislocalisation underlies this effect. Together, these findings demonstrate that FACT facilitates cohesin translocation along chromatin, promoting efficient loop extrusion while preventing its aberrant accumulation within transcribed regions. This work provides mechanistic insight into how chromatin structure modulates cohesin function to coordinate genome organisation with transcription in mammalian cells.

Nucleosomes regulate DNA accessibility and thereby influence gene expression, DNA repair, and mutagenesis. Although DNA sequence strongly determines nucleosome positioning, the higher-order sequence grammar underlying nucleosome organization and its relationship to cancer-associated mutational and epigenetic processes remain poorly understood. We developed DeepND, an interpretable deep learning framework that distinguishes nucleosomal from inter-nucleosomal DNA and identifies sequence features associated with nucleosome architecture. Our interpretable framework enabled analysis of sequence patterns and identified 210 sequence motifs of 2-16 bp length that were significantly enriched or depleted in nucleosomal DNA, substantially extending the previously characterized nucleosomal motifs. These motifs revealed distinct sequence grammars within nucleosomal footprints and linker regions, including patterns associated with inward- and outward-facing minor grooves. Forty-four of these motifs showed cancer-type-specific enrichment or depletion of somatic mutations, and six motifs were associated with differential DNA methylation levels. In lung and endometrial cancers, mutations exhibited a significant ~10-bp periodic enrichment pattern across distinct minor-groove orientations, consistent with differential sensitivity of mutational and DNA repair processes to nucleosomal rotational positioning. We further identified sequence-specific methylation patterns consistent with reduced accessibility of nucleosomal DNA to DNA methyltransferases, including differential methylation at inward- and outward-facing CpG motifs. Together, these findings provide a sequence-resolved characterization of nucleosome-level organization and its link to cancer-specific mutational processes and epigenetic landscapes.

Centromere identity is specified epigenetically by nucleosomes containing the histone H3 variant CENP-A, yet centromeres are almost invariably embedded within repetitive DNA, suggesting that underlying DNA sequences contribute to the organization of centromeric chromatin. In Drosophila melanogaster, CENP-A primarily occupies retroelement-rich islands flanked by simple satellite DNA, but their functional contribution remains unclear. Here, we delete the ~50-kb island Giglio from centromere 3 while preserving the flanking satellites and residual CENP-A chromatin. Despite removal of the DNA sequence associated with most centromeric CENP-A, centromere identity and overall CENP-A levels are preserved at the native locus, indicating that the island itself is not required for centromere identity. Instead, Giglio deletion increases variability in CENP-A chromatin organization, producing highly heterogeneous domain sizes and preferential redistribution of the CENP-A domain into the adjacent dodeca satellite. Together, these findings demonstrate that Giglio is dispensable for centromere identity but constrains the size and position of the CENP-A domain, establishing centromeric islands as key determinants of CENP-A domain architecture.

Heterochromatin loss disrupts nuclear architecture, gene regulation and repetitive element silencing, and is associated with diverse human diseases. However, mechanisms linking heterochromatin dysfunction to pathological phenotypes remain unclear. Using genetic interaction screening and genomic analyses in C. elegans, we identify secondary activation of the Intracellular Pathogen Response (IPR), an innate immune stress pathway, as a major contributor to heterochromatin mutant phenotypes. Constitutive IPR activation phenocopies slow growth and indirect transcriptional changes observed in these mutants. Depletion of genetic enhancers further increased, whereas suppressor RNAi attenuated IPR activation, with direct heterochromatin targets remaining substantially deregulated. Notably, many suppressors encode active chromatin components, and mild reduction of RNA polymerase II activity ameliorates growth defects in C. elegans HP1 mutants and human HP1-deficient cells. Our findings reveal secondary stress response activation as an important mechanism linking heterochromatin dysfunction to pathology and identify transcriptional dampening as a potential therapeutic strategy for mitigating these effects.

Recurrent pregnancy loss (RPL) affects 5% of couples worldwide. Over half of cases remain unexplained (uRPL), in part because paternal factors are rarely evaluated beyond paternal karyotype. Sperm DNA methylation is a putative paternal contributor to RPL. Using ONT direct whole methylome sequencing, we generated the first and largest cohort of locus-resolved, single-molecule sperm methylome maps from men with uRPL to date (n=39). We identified 294 differentially methylated regions (DMRs), of which 158 (53.7%) overlapped AluY repetitive elements, representing significant enrichment (z=40.9); most were hypomethylated in uRPL sperm and spanned the full length of individual AluY insertions. Because of single-molecule resolution, we show that the methylation differences found in uRPL sperm occur in a subpopulation of sperm rather than a uniform DNA methylation change across all sperm. Affected loci were underrepresented for imprinting-associated chromatin marks and instead overrepresented for H2A.Z. Although DMR-associated genes were enriched for neuronal GO terms, many converge on reported roles in implantation, placentation, and early embryonic development. AluY epimutation represents a previously unrecognized, predominant signature of paternal epigenetic perturbation in uRPL, establishing repetitive elements as a new axis of sperm epigenetic risk.

Prospero-related homeobox (Prox) genes encode a family of transcription factors that play essential roles in the development of several organs and systems, including the central nervous system, lymphatic endothelium, musculature, and liver. Despite their developmental importance, the evolutionary history of the vertebrate Prox gene family remains poorly understood. In this study we combined phylogenetic and synteny analysis to characterise the evolution of the Prox family in vertebrates. Our results reveal that two to three Prox subfamilies were already present in the last common ancestor of jawed vertebrates. We clarify the identity and evolutionary relationships of well-studied members of this family and identify multiple independent losses of Prox2 and Prox3 genes in specific vertebrate lineages. Furthermore, we uncover evidence for the existence of a fourth Prox gene in the ancestral vertebrate genome, which was subsequently lost. Overall, this study provides the first comprehensive analysis of the evolutionary history of the vertebrate Prox gene family and establishes a foundations for future studies on the functional roles of these genes.

Transposable elements (TEs) are a major source of genomic diversity in mammals, yet their regulatory roles in the bovine genome remain poorly understood. Through characterizing bovine TE landscape, despite the substantial proportion (25.6%) of ruminant-specific TEs, we observe age- and class-dependent genomic distribution patterns similar to those observed in other mammals. Next, we profile TE and gene expression dynamics in pre-implantation embryos generated in vivo (IVV), by in vitro fertilization (IVT), and through somatic cell nuclear transfer (SCNT). The zygotic genome activation (ZGA) is shifted from the 4-cell stage to the 8-cell stage in IVT and SCNT embryos compared to IVV embryos. SCNT embryos exhibit impaired initiation of early transcription programs at the 4-cell stage and disrupted developmental trajectories, including abnormal activation of pluripotency-associated genes. A subset of retroviral LTR elements are strongly activated at ZGA in IVV and IVT embryos, whereas their activation is markedly muted in SCNT embryos, suggesting that impaired gene and TE reprogramming may contribute to the developmental defects commonly observed in SCNT embryos. By epigenomic profiling, the MLT1D elements from the ERVL-MaLR LTR family lose repressive marks and gain H3K27ac at ZGA, together with DUXA-binding motif enrichment. Knockdown of DUXA in bovine embryos significantly reduced MLT1D expression and ZGA marker genes. We propose that a subset of DUXA-enriched MLT1D functions as enhancers that promote ZGA. Overall, our study provides new insights into the regulatory roles of TEs during bovine embryogenesis and establishes a framework for comparative studies of TE-mediated gene regulation in early mammalian development.

Chromosome was "self-assembled" into a complex three-dimensional topology in the nucleus, which provided the structural basis for important biological processes such as gene expression regulation and cell differentiation. The current understanding of the three-dimensional organization of the genome is based on high-throughput sequencing and imaging technologies to visualize the conformation of chromatin in its native environment. This study proposes a new research perspective and technical means to use fusion genes to determine the interaction information and three-dimensional structure of chromosomes. This three-dimensional information provided by the fusion gene spectrum not only shows the universality of the spatial layout of chromosomes, but also increases the interpretability of the occurrence of fusion genes. The chromatin conformation capture data of multiple tissues and periods is significantly correlated with the strength of fusion genes. For example, the frequency of trans-interaction between chromosomes 11 and 17 was the highest, and the frequency of fusion gene occurrence corresponding to KMT2A on chromosome 11 was the highest. Therefore, we infer that this structural universality is a common feature of the human genome, which provides a theoretical basis and data for predicting chromosome fusion events.

JAK-STAT signalling pathway activation relies mostly on the transcriptional regulation of its ligands. The pathway, conserved in vertebrates and invertebrates, is required for the development and homeostasis of various organs. In contrast to the complexity of the vertebrate pathway, with a multiplicity of ligands, receptors, kinases and transcription factors, the simplicity of the Drosophila pathway with only three ligands (Upd1, Upd2 and Upd3), a single receptor (Dome), a JAK kinase (Hop) and a STAT transcription factor (STAT92E) facilitates the genetic study of its multiple functions. To discover how, when and where the Upd1 ligand performs each organogenetic function, we have searched for the different enhancers controlling its dynamic spatio-temporal regulation and used them to generate minigenes replicating specific stages of Upd1 expression. The combination of these minigenes with mutations deleting all three Upd ligands, allowed us to identify the timing when the various embryonic functions are mediated, including head involution, tracheae, corpora allata and prothoracic gland specification, posterior spiracle formation and hindgut elongation. We uncover most of the enhancers controlling STAT activation during embryonic organogenesis and those driving activation in the adult testis and the ovary. As expected, we identified redundant shadow enhancers but, interestingly, we found a different type of redundant enhancers that mediate identical developmental functions although driving completely different expression patterns. We name this new class of functionally redundant enhancers driving different expression as shade enhancers. We propose shade enhancers provide evolutionary plasticity as they could allow the pattern of gene expression to diversify without altering the underlying body plan.

In the germline, small RNA-mediated gene regulatory pathways are associated with phase separated condensates known as germ granules across species. Germ granules can be challenging to purify based on their biophysical properties, therefore we used an in vivo proximity labeling approach, TurboID, to define the proximal interaction networks of the germ granule-localized Argonaute proteins: CSR-1, PRG-1/Piwi, PPW-2/WAGO-3, WAGO-1 and WAGO-4, and the nuclear-enriched Argonaute HRDE-1 as a comparator, in C. elegans. We found that although CSR-1 and WAGO-4 bind to the same set of 22G- small RNAs and target an overlapping set of germline genes, they possess distinct proximal interaction networks. Displacement of WAGO-4 from the germ granules highlights distinct interactors involved in chromosome biology and points to a potential nuclear role for WAGO-4. PRG-1/Piwi, WAGO-1, and PPW-2/WAGO-3, act in a common pathway and share an interaction network, while HRDE-1 displays a distinct interaction network that highlights its potential to influence splicing, rather than its widely accepted role in histone modification. Performing TurboID on other sRNA pathway factors, including the RdRP RRF-1 led us to uncover a potential nuclear role for this protein, and overlapping our TurboID data with published proximity labeling data enabled us to identify a core set of eleven mostly uncharacterized proteins that play roles in fertility, germ granule organization, and transgenerational epigenetic inheritance. One of these factors, D2005.4, appears to be a heme-binding germ granule-localized protein with a Tetratricopeptide Repeat Domain that interacts with all germ granule AGOs and may play scaffolding or enzymatic roles in germ granules. Collectively, our work highlights the utility of proximity labelling to uncover insights into AGO and sRNA pathway factor function and identify new factors that could be of importance in germ cell biology.

DNA polymerases generally interact with the processivity clamp (DnaN/{beta}-clamp) during replication via clamp-binding motifs (CBMs). Since a CBM was not detected in DNA Polymerase I (PolI) from E. coli, it is generally believed that PolI performs its functions in genome replication and repair, independently of the {beta}-clamp. We observe that, while PolI and {beta}-clamp proteins from E. coli do not interact, their homologs from Mycobacterium tuberculosis bind strongly, and the binding site localises to the Klenow fragment of PolI. The cryo-EM structure (4.9 [A]) of the Mtb-PolI-Klenow: Mtb-{beta}-clamp complex, coupled with continuous heterogeneity analysis of the cryo-EM data and binding experiments, showed that the primary CBM in PolI may be defined by the stretch 457QLSLLDD463. The crystal structure (2.9 [A]) of the {beta}-clamp with the CBM peptide revealed specific interactions that stabilise the peptide within the {beta}-clamp's canonical binding site. Phylogenetic analyses across different bacteria showed that the identified CBM is present only in PolI from members of the order Mycobacteriales. Overall, the interaction between PolI with the {beta}-clamp, mediated by a CBM unique to the Mycobacteriales order, highlights the mechanistic distinctiveness of genome replication in Mtb and phylogenetically related organisms, and has important implications for drug discovery.

RNA-binding proteins (RBPs) are increasingly recognized to perform chromatin-associated functions in addition to their canonical roles in RNA regulation, yet how these activities are coordinated in human embryonic stem cells (hESCs) remain poorly understood. Here, we characterize distinct RNA- and chromatin-associated activities for RBM25 in hESCs. Transcriptome-wide profiling showed that RBM25 preferentially associates with transcripts involved in RNA processing and splicing, consistent with its canonical role as an RBP involved in post-transcriptional regulation. Genome-wide chromatin profiling further uncovered a distinct chromatin-associated program, with RBM25 occupying accessible promoters of actively expressed cell-cycle genes and introns predominantly associated with transcriptionally inactive neuronal genes. Interestingly, RBM25 chromatin-binding sites showed substantial co-occupancy with regions bound by the transcription factor CEBPZ in hESCs. We further show that RBM25 and CEBPZ physically interact and exhibit shared chromatin-binding patterns at both promoter and intronic regions. Importantly, reciprocal depletion experiments revealed that RBM25 is required for CEBPZ occupancy at CCAAT-containing promoters of highly expressed cell-cycle genes, whereas CEBPZ is not similarly required for RBM25 chromatin occupancy. Together, our findings establish RBM25 as a multifunctional factor with distinct roles in RNA processing and chromatin-associated regulation and reveal a context-dependent RBP-TF interaction that contributes to transcriptional regulation in hESCs.

Embryonic ovarian development requires high levels of WNT/B-catenin signaling yet the molecular basis of this requirement remains unclear. Mutations in the WNT potentiator RSPO1 impair ovarian development in both mouse and human, leading to gonadal masculinization in XX individuals. Here, we show that RSPO1 acts within a restricted developmental window at the onset of ovarian differentiation and that its absence triggers precocious androgen signaling in the developing ovaries. Moreover, androgen receptor inhibition rescues ovarian development in Rspo1 mutant mice. Our findings identify RSPO1/WNT/B-catenin signaling as an essential pathway that suppresses androgen signaling to ensure proper ovarian development in mouse.

Background: X-chromosome inactivation (XCI) evolved independently in eutherian and marsupial mammals, where it is orchestrated by the unrelated long non-coding RNAs Xist and RSX, respectively. Xist organizes a repressive nuclear compartment through multivalent RNA-protein interactions, but whether RSX exploits similar biophysical principles remains unknown. A recent paper has identified bona fide RSX interacting proteins. Results: We integrated proteome-scale RNA-protein interaction prediction, experimental validation, phase-separation propensity analysis, functional annotation and comparative RNA-structure modelling to characterize the RSX interaction landscape. Using catRAPID, we ranked 1,168 RNA-binding proteins from the native Monodelphis domestica proteome. Predictions were significantly enriched for experimentally identified RSX interactors, with 4.85-fold enrichment among the top 50 candidates (P approximately 1.6 x 10-5), increasing to approximately eightfold for proteins shared by the experimental RSX and Xist interactomes (P approximately 2 x 10-6). Among 30 high-confidence RSX interactors, 13 were experimentally supported, 17 were previously unrecognized candidates and 17 exhibited high phase-separation propensity. The network was enriched in ribonucleoprotein granules and nuclear bodies and converged on m6A regulators and SR-family splicing factors. Comparative modelling detected no conserved secondary or tertiary architecture between RSX and Xist. Conclusions: RSX and Xist appear to have converged not through RNA sequence or global structure, but through recruitment of related, condensation-prone protein networks. These findings identify interaction-network and biophysical convergence as a potential principle of lncRNA-mediated chromosome regulation and provide testable candidates for determining whether RSX establishes a condensate-like compartment on the marsupial inactive X.

Palmitate-associated lipotoxic stress activates the endoplasmic-reticulum stress sensor protein IRE1, but how IRE1 signaling influences genotoxic-stress responses in triple-negative breast cancer remains unclear. Here, we investigated IRE1 and XBP1 signaling in MDA-MB-231 cells exposed to palmitate and etoposide. Under combined lipotoxic and genotoxic stress, XBP1 depletion increased DNA-damage accumulation in IRE1 wild-type cells but not in IRE1-deficient cells, whereas IRE1 re-expression reduced DNA damage. Pharmacologic IRE1 RNase inhibition also increased etoposide-associated DNA damage, supporting a protective role for IRE1 RNase-dependent signaling. IRE1 status and XBP1 manipulation differentially affected H2A.X and {gamma}H2A.X abundance, indicating context-dependent regulation of chromatin-associated DNA-damage signaling. At the RNA level, H2A.X was enriched in IRE1 immunoprecipitates, and the H2A.X-linked DNA-damage-response transcripts HUWE1 and BRCA2 also associated with IRE1 under combined stress. These findings link palmitate-activated IRE1-XBP1 signaling to genome-protective responses during genotoxic stress and identify a network of IRE1-associated DNA-damage-response transcripts in triple-negative breast cancer cells. Direct IRE1-mediated cleavage of these transcripts remains to be established.

Removal of TE-like DNA methylation from regulatory elements drives canonical genomic imprinting of both maternally and paternally expressed genes in endosperm (MEGs and PEGs). Here we report that removal of CG-only gene body methylation also drives imprinting in maize, resulting in maternally repressed PEGs. These CG body methylated PEGs express broadly across cell types and lack H3K27me3 except in endosperm, where maternal copies acquire it. We found that partial gene body demethylation is a widespread feature of endosperm genes, usually does not result in imprinted expression, and is mediated by DNA glycosylases/lyases (DNGs). These and other data indicate that gene body methylation can shield select genes from PRC2 repression, and its maternal removal by DNGs explains the majority of PEGs in maize and rice but has weak impact in Arabidopsis thaliana. We hypothesize that increased gene expression noise arising from gene body demethylation--both with and without imprinting--is beneficial in endosperm.

Temperature influences fertility across diverse organisms, yet the mechanisms underlying how suboptimal temperatures affect gamete production and quality remain largely unknown. We previously showed that chronic exposure of adult Drosophila melanogaster females to mild cold promotes the maintenance of germline stem cells (GSCs) and high oocyte quality over time despite reducing the rates of oogenesis, while exposure to warm temperature causes death of early germline cysts and vitellogenic follicles and a severe decrease in oocyte quality. To explore potential mechanisms underlying these highly distinct responses, we compared the ovarian transcriptomes of females maintained at these temperatures (18{degrees}C or 29{degrees}C) to that of 25{degrees}C controls. We found that 18{degrees}C upregulates or downregulates ~2.5 times as many genes as 29{degrees}C, indicating that the ovary mounts active physiological responses to mild cold and warm temperatures--as opposed to simply undergoing passive changes driven by thermodynamics. Gene set enrichment analysis revealed modulation of genes involved in neuronal signaling in opposite directions at 18{degrees}C versus 29{degrees}C. Most genes, however, exhibit temperature-specific regulation: 29{degrees}C upregulates synaptic transmission genes and downregulates lipid biosynthesis genes, whereas 18{degrees}C upregulates actin cytoskeleton genes and downregulates cell adhesion and lipid organization genes. Notably, mild cold or warm temperature specifically modulated (either up or down) the expression of distinct sets of transposable elements (TEs), suggesting the existence of temperature-dependent TE regulatory mechanisms and/or downstream effects. Finally, we show that GSCs at 18{degrees}C have increased retrotransposon R2 transcript levels, larger nucleolar size, and elevated levels of the known stemness factor phosphorylated Mad, leading to a working model whereby elevated ribosome biogenesis supports increased stemness signaling to promote GSC maintenance in mild cold. These findings suggest potential mechanisms and open new questions for investigation towards a deeper understanding of how temperature modulates gene expression and impacts germline development and quality--which are essential for the perpetuation of species.