Designing realistic regulatory DNA with autoregressive language models.

Cis-regulatory elements (CREs), such as promoters and enhancers, are DNA sequences that regulate the expression of genes. The activity of a CRE is influenced by the order, composition, and spacing of sequence motifs that are bound by proteins called transcription factors (TFs). Synthetic CREs with specific properties are needed for biomanufacturing as well as for many therapeutic applications including cell and gene therapy. Here, we present regLM, a framework to design synthetic CREs with desired properties, such as high, low, or cell type-specific activity, using autoregressive language models in conjunction with supervised sequence-to-function models. We used our framework to design synthetic yeast promoters and cell type-specific human enhancers. We demonstrate that the synthetic CREs generated by our approach are not only predicted to have the desired functionality but also contain biological features similar to experimentally validated CREs. regLM thus facilitates the design of realistic regulatory DNA elements while providing insights into the cis-regulatory code.

Cis-acting variation is common across regulatory layers but is often buffered during embryonic development.

Precise patterns of gene expression are driven by interactions between transcription factors, regulatory DNA sequences, and chromatin. How DNA mutations affecting any one of these regulatory "layers" are buffered or propagated to gene expression remains unclear. To address this, we quantified allele-specific changes in chromatin accessibility, histone modifications, and gene expression in F1 embryos generated from eight Drosophila crosses at three embryonic stages, yielding a comprehensive data set of 240 samples spanning multiple regulatory layers. Genetic variation (allelic imbalance) impacts gene expression more frequently than chromatin features, with metabolic and environmental response genes being most often affected. Allelic imbalance in cis-regulatory elements (enhancers) is common and highly heritable, yet its functional impact does not generally propagate to gene expression. When it does, genetic variation impacts RNA levels through two alternative mechanisms involving either H3K4me3 or chromatin accessibility and H3K27ac. Changes in RNA are more predictive of variation in H3K4me3 than vice versa, suggesting a role for H3K4me3 downstream from transcription. The impact of a substantial proportion of genetic variation is consistent across embryonic stages, with 50% of allelic imbalanced features at one stage being also imbalanced at subsequent developmental stages. Crucially, buffering, as well as the magnitude and evolutionary impact of genetic variants, is influenced by regulatory complexity (i.e., number of enhancers regulating a gene), with transcription factors being most robust to cis-acting, but most influenced by trans-acting, variation.

The cis-regulatory dynamics of embryonic development at single-cell resolution.

Understanding how gene regulatory networks control the progressive restriction of cell fates is a long-standing challenge. Recent advances in measuring gene expression in single cells are providing new insights into lineage commitment. However, the regulatory events underlying these changes remain unclear. Here we investigate the dynamics of chromatin regulatory landscapes during embryogenesis at single-cell resolution. Using single-cell combinatorial indexing assay for transposase accessible chromatin with sequencing (sci-ATAC-seq), we profiled chromatin accessibility in over 20,000 single nuclei from fixed Drosophila melanogaster embryos spanning three landmark embryonic stages: 2-4 h after egg laying (predominantly stage 5 blastoderm nuclei), when each embryo comprises around 6,000 multipotent cells; 6-8 h after egg laying (predominantly stage 10-11), to capture a midpoint in embryonic development when major lineages in the mesoderm and ectoderm are specified; and 10-12 h after egg laying (predominantly stage 13), when each of the embryo's more than 20,000 cells are undergoing terminal differentiation. Our results show that there is spatial heterogeneity in the accessibility of the regulatory genome before gastrulation, a feature that aligns with future cell fate, and that nuclei can be temporally ordered along developmental trajectories. During mid-embryogenesis, tissue granularity emerges such that individual cell types can be inferred by their chromatin accessibility while maintaining a signature of their germ layer of origin. Analysis of the data reveals overlapping usage of regulatory elements between cells of the endoderm and non-myogenic mesoderm, suggesting a common developmental program that is reminiscent of the mesendoderm lineage in other species. We identify 30,075 distal regulatory elements that exhibit tissue-specific accessibility. We validated the germ-layer specificity of a subset of these predicted enhancers in transgenic embryos, achieving an accuracy of 90%. Overall, our results demonstrate the power of shotgun single-cell profiling of embryos to resolve dynamic changes in the chromatin landscape during development, and to uncover the cis-regulatory programs of metazoan germ layers and cell types.

An otopetrin family proton channel promotes cellular acid efflux critical for biomineralization in a marine calcifier.

Otopetrins comprise a family of proton-selective channels that are critically important for the mineralization of otoliths and statoconia in vertebrates but whose underlying cellular mechanisms remain largely unknown. Here, we demonstrate that otopetrins are critically involved in the calcification process by providing an exit route for protons liberated by the formation of CaCO3 Using the sea urchin larva, we examined the otopetrin ortholog otop2l, which is exclusively expressed in the calcifying primary mesenchymal cells (PMCs) that generate the calcitic larval skeleton. otop2l expression is stimulated during skeletogenesis, and knockdown of otop2l impairs spicule formation. Intracellular pH measurements demonstrated Zn2+-sensitive H+ fluxes in PMCs that regulate intracellular pH in a Na+/HCO3--independent manner, while Otop2l knockdown reduced membrane proton permeability. Furthermore, Otop2l displays unique features, including strong activation by high extracellular pH (>8.0) and check-valve-like outwardly rectifying H+ flux properties, making it into a cellular proton extrusion machine adapted to oceanic living conditions. Our results provide evidence that otopetrin family proton channels are a central component of the cellular pH regulatory machinery in biomineralizing cells. Their ubiquitous occurrence in calcifying systems across the animal kingdom suggest a conserved physiological function by mediating pH at the site of mineralization. This important role of otopetrin family proton channels has strong implications for our view on the cellular mechanisms of biomineralization and their response to changes in oceanic pH.

Genetic variants regulating expression levels and isoform diversity during embryogenesis.

Embryonic development is driven by tightly regulated patterns of gene expression, despite extensive genetic variation among individuals. Studies of expression quantitative trait loci (eQTL) indicate that genetic variation frequently alters gene expression in cell-culture models and differentiated tissues. However, the extent and types of genetic variation impacting embryonic gene expression, and their interactions with developmental programs, remain largely unknown. Here we assessed the effect of genetic variation on transcriptional (expression levels) and post-transcriptional (3' RNA processing) regulation across multiple stages of metazoan development, using 80 inbred Drosophila wild isolates, identifying thousands of developmental-stage-specific and shared QTL. Given the small blocks of linkage disequilibrium in Drosophila, we obtain near base-pair resolution, resolving causal mutations in developmental enhancers, validated transcription-factor-binding sites and RNA motifs. This fine-grain mapping uncovered extensive allelic interactions within enhancers that have opposite effects, thereby buffering their impact on enhancer activity. QTL affecting 3' RNA processing identify new functional motifs leading to transcript isoform diversity and changes in the lengths of 3' untranslated regions. These results highlight how developmental stage influences the effects of genetic variation and uncover multiple mechanisms that regulate and buffer expression variation during embryogenesis.

Can Clinically Node-Negative Breast Cancer Patients with Suspicious Axillary Lymph Nodes at Ultrasound But Negative Fine-Needle Aspiration Be Approached as Having Node-Negative Disease?

BACKGROUND: Few data exist to elucidate whether patients with a suspicious axillary lymph node (ALN) at ultrasound but a negative fine-needle aspiration result (FNA group) can be managed as having ultrasound node-negative disease (AUN group). This study compared various ALN statuses between the AUN and FNA groups to guide further ALN management. METHODS: Patients who had clinical T1-2N0 breast cancer treated with sentinel lymph node (SLN) biopsy were retrospectively analyzed. The ALN metastasis status, SLN status, and non-SLN metastasis rates in the entire population and the patients with one or two positive SLNs were compared between the AUN and FNA groups. RESULTS: A total of 1007 patients (886 AUN and 121 FNA patients) were eligible for the final analysis: The incidence of ALN metastasis did not differ between the AUN group (16.5%) and the FNA group (21.5%) (P = 0.170). In addition, three or more metastases were found in only 2.4% of the AUN patients and 3.3% of the FNA patients (P = 0.405). The non-SLN metastasis rate was 22.6% (33/146) in the AUN group and 19.2% (5/26) in the FNA group (P = 0.699). For the patients with one or two positive SLNs, the rate of non-SLN metastasis was similar between the AUN group (19.6%, 27/138) and the FNA group (12.5%, 3/24) (P = 0.591). CONCLUSIONS: Patients with a suspicious ALN at ultrasound but a negative FNA result had ALN statuses similar to those of the ultrasound node-negative patients, indicating that these patients can be treated as having ultrasound node-negative disease.

Next-generation sequencing-based detection of germline L1-mediated transductions.

BACKGROUND: While active LINE-1 (L1) elements possess the ability to mobilize flanking sequences to different genomic loci through a process termed transduction influencing genomic content and structure, an approach for detecting polymorphic germline non-reference transductions in massively-parallel sequencing data has been lacking. RESULTS: Here we present the computational approach TIGER (Transduction Inference in GERmline genomes), enabling the discovery of non-reference L1-mediated transductions by combining L1 discovery with detection of unique insertion sequences and detailed characterization of insertion sites. We employed TIGER to characterize polymorphic transductions in fifteen genomes from non-human primate species (chimpanzee, orangutan and rhesus macaque), as well as in a human genome. We achieved high accuracy as confirmed by PCR and two single molecule DNA sequencing techniques, and uncovered differences in relative rates of transduction between primate species. CONCLUSIONS: By enabling detection of polymorphic transductions, TIGER makes this form of relevant structural variation amenable for population and personal genome analysis.

Impact of genomic structural variation in Drosophila melanogaster based on population-scale sequencing.

Genomic structural variation (SV) is a major determinant for phenotypic variation. Although it has been extensively studied in humans, the nucleotide resolution structure of SVs within the widely used model organism Drosophila remains unknown. We report a highly accurate, densely validated map of unbalanced SVs comprising 8962 deletions and 916 tandem duplications in 39 lines derived from short-read DNA sequencing in a natural population (the "Drosophila melanogaster Genetic Reference Panel," DGRP). Most SVs (>90%) were inferred at nucleotide resolution, and a large fraction was genotyped across all samples. Comprehensive analyses of SV formation mechanisms using the short-read data revealed an abundance of SVs formed by mobile element and nonhomologous end-joining-mediated rearrangements, and clustering of variants into SV hotspots. We further observed a strong depletion of SVs overlapping genes, which, along with population genetics analyses, suggests that these SVs are often deleterious. We inferred several gene fusion events also highlighting the potential role of SVs in the generation of novel protein products. Expression quantitative trait locus (eQTL) mapping revealed the functional impact of our high-resolution SV map, with quantifiable effects at >100 genic loci. Our map represents a resource for population-level studies of SVs in an important model organism.

The impact of gene expression variation on the robustness and evolvability of a developmental gene regulatory network.

Regulatory interactions buffer development against genetic and environmental perturbations, but adaptation requires phenotypes to change. We investigated the relationship between robustness and evolvability within the gene regulatory network underlying development of the larval skeleton in the sea urchin Strongylocentrotus purpuratus. We find extensive variation in gene expression in this network throughout development in a natural population, some of which has a heritable genetic basis. Switch-like regulatory interactions predominate during early development, buffer expression variation, and may promote the accumulation of cryptic genetic variation affecting early stages. Regulatory interactions during later development are typically more sensitive (linear), allowing variation in expression to affect downstream target genes. Variation in skeletal morphology is associated primarily with expression variation of a few, primarily structural, genes at terminal positions within the network. These results indicate that the position and properties of gene interactions within a network can have important evolutionary consequences independent of their immediate regulatory role.

Medically induced euthyroid hypothyroxinemia may extend survival in compassionate need cancer patients: an observational study.

BACKGROUND: Clinical studies have shown that interventional lowering of serum free thyroxine (FT4) may be associated with extended survival in patients with some terminal cancers. The report of success with this approach in glioblastoma multiforme caused involvement of the author (A.H.) in the prospective consultative management of 23 end-stage solid tumor patients in whom hypothyroxinemia was induced to prolong life. PATIENTS AND METHODS: Patients were self-referred or recommended by attending physicians to the author (A.H.) and had advanced cancers of the brain, ovary, lung, pancreas, salivary gland, and breast or had mesothelioma or soft-tissue sarcoma. Hypothyroxinemia was achieved in euthyroid patients by using methimazole, with the addition of 3,3',5-triiodo-L-thyronine (L-T3) to prevent hypothyroidism and suppress endogenous thyrotropin (TSH). In patients with pre-existent primary hypothyroidism, T3 administration was substituted for T4 replacement. Serum FT4 and TSH concentrations were serially monitored to enable adjustments to drug therapy and prevent clinical hypothyroidism. Survival was measured from the date of hypothyroxinemia induction with T3 or methimazole plus T3. Outcomes were compared with the odds of death based on the Surveillance Epidemiology and End Results and American Joint Committee on Cancer databases and literature reports. RESULTS: The survival time of 83% (19 of 23) of patients exceeded the 20% expected 1-year survival for this hypothyroxinemic, end-stage cancer group. The difference between actual and expected survival was significant. CONCLUSION: Although this is an uncontrolled observational experience with frank limitations, compassionate medical induction of hypothyroxinemia should be considered for patients with advanced cancers to whom other avenues of treatment are closed.

Ultrasensitive proteome analysis using paramagnetic bead technology.

In order to obtain a systems-level understanding of a complex biological system, detailed proteome information is essential. Despite great progress in proteomics technologies, thorough interrogation of the proteome from quantity-limited biological samples is hampered by inefficiencies during processing. To address these challenges, here we introduce a novel protocol using paramagnetic beads, termed Single-Pot Solid-Phase-enhanced Sample Preparation (SP3). SP3 provides a rapid and unbiased means of proteomic sample preparation in a single tube that facilitates ultrasensitive analysis by outperforming existing protocols in terms of efficiency, scalability, speed, throughput, and flexibility. To illustrate these benefits, characterization of 1,000 HeLa cells and single Drosophila embryos is used to establish that SP3 provides an enhanced platform for profiling proteomes derived from sub-microgram amounts of material. These data present a first view of developmental stage-specific proteome dynamics in Drosophila at a single-embryo resolution, permitting characterization of inter-individual expression variation. Together, the findings of this work position SP3 as a superior protocol that facilitates exciting new directions in multiple areas of proteomics ranging from developmental biology to clinical applications.

Surgery time interval and molecular subtype may influence Ki67 change after core needle biopsy in breast cancer patients.

BACKGROUND: To investigate the accuracy of core needle biopsy (CNB) in evaluating breast cancer estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 status and to identify factors which might be associated with Ki67 value change after CNB. METHODS: A retrospective study was carried out on 276 patients with paired CNB and surgically removed samples (SRS). Clinico-pathological factors as well as the surgery time interval (STI) between CNB and surgery were analyzed to determine whether there were factors associated with Ki67 value change after CNB. Five tumor subtypes were classified as follows: Luminal A, Luminal B-HER2-, Luminal B-HER2+, Triple Negative (TN), and HER2+. Ki67 value change was calculated as SRS minus CNB. RESULTS: Mean STI after CNB was 4.5 (1-37) days. Good agreement was achieved for ER, PR, and HER2 evaluation between CNB and SRS. However, Ki67 expression level was significantly higher in SRS compared with CNB samples: 29.1 % vs. 26.2 % (P < 0.001). Both univariate and multivariate analysis demonstrated that STI and molecular subtype were associated with a Ki67 change after CNB. Luminal A tumors experienced more Ki67 elevation than Luminal B-HER2- diseases (6.2 % vs -0.1 %, P = 0.014). Patients with longer STI after CNB had a higher Ki67 increase: -1.1 % within 1-2 days, 2.1 % with 3-4 days, and 5.6 % more than 4 days, respectively (P = 0.007). For TN and HER2+ tumors, the Ki67 change was apt to be 0 with STI ≤ 4 days, while a >7 % Ki67 increase was noticed in patients with STI ≥ 5 days. CONCLUSION: CNB was accurate in evaluating ER, PR, HER2, and molecular subtype status. Ki67 value significantly increased after CNB, which was associated with STI and molecular subtype. Further translational research needs to consider Ki67 changes following CNB among different breast cancer molecular subtypes.

Traumatic brain injury, coronary atherosclerosis and cardiovascular mortality.

BACKGROUND: Traumatic-brain-injury (TBI) is a devastating-condition resulting in cerebral edema and ischemia. This study investigates the association of mild-TBI (mTBI) to sub-clinical atherosclerosis and cardiovascular (CV) mortality. METHODS: Five hundred and forty-three veterans without known coronary artery disease or diagnosed mental disorder, who underwent coronary artery calcium (CAC) scanning for clinical indications, were followed for a median of 4-years. Veterans' medical diagnoses and neuropsychiatric health status (mTBI vs non-mTBI) were evaluated using VA electronic medical records. CAC was defined as 0, 1-100, 101-400 and 400+. RESULTS: CAC was higher in mTBI, compared to without-mTBI (p < 0.05). TBI was more prevalent with the-severity of CAC (p < 0.05). Regression-analyses revealed that mTBI is an independent-predictor of CAC (p < 0.01). The CV mortality rate was 25% in mTBI and 10.5% in without-mTBI (p = 0.0001). Multivariable survival regression analyses revealed a significant-association between mTBI and CAC, with increased-risk of CV mortality (p < 0.05). The hazard-ratio of CV mortality was 5.25 in mTBI & CAC > 0, compared to without-mTBI & CAC = 0 (p < 0.05). The risk of CV-mortality was 2.25 for mTBI & CAC = 1-100, 4.93 for mTBI & CAC = 101-400 and 7.06 for mTBI & CAC ≥ 400, compared to matched CAC-categories without-mTBI (p < 0.05). The area under ROC curve to predict CV mortality was 0.64 for mTBI, 0.69 for mTBI & PTSD, 0.85 for mTBI & CAC > 0 and 0.92 for the combination. The prognostication of mTBI to predict CV mortality is superior to the Framingham risk score. Also the combination of mTBI & PTSD provided incremental prognostic values to predict CV mortality (p < 0.05). CONCLUSIONS: mTBI is associated with the severity of sub-clinical coronary atherosclerosis and independently predicts CV mortality.

Stromal cells in tumor microenvironment and breast cancer.

Cancer is a systemic disease encompassing multiple components of both tumor cells themselves and host stromal cells. It is now clear that stromal cells in the tumor microenvironment play an important role in cancer development. Molecular events through which reactive stromal cells affect cancer cells can be defined so that biomarkers and therapeutic targets can be identified. Cancer-associated fibroblasts (CAFs) make up the bulk of cancer stroma and affect the tumor microenvironment such that they promote cancer initiation, angiogenesis, invasion, and metastasis. In breast cancer, CAFs not only promote tumor progression but also induce therapeutic resistance. Accordingly, targeting CAFs provides a novel way to control tumors with therapeutic resistance. This review summarizes the current understandings of tumor stroma in breast cancer with a particular emphasis on the role of CAFs and the therapeutic implications of CAFs. In addition, the effects of other stromal components such as endothelial cells, macrophages, and adipocytes in breast cancer are also discussed. Finally, we describe the biologic markers to categorize patients into a specific and confirmed subtype for personalized treatment.

PD-1(+) immune cell infiltration inversely correlates with survival of operable breast cancer patients.

The programmed death-1 (PD-1) molecule is mainly expressed on functionally "exhausted" CD8(+) T cells, dampening the host antitumor immune response. We evaluated the ratio between effective and regulatory T cells (Tregs) and PD-1 expression as a prognostic factor for operable breast cancer patients. A series of 218 newly diagnosed invasive breast cancer patients who had undergone primary surgery at Ruijin Hospital were identified. The influence of CD8(+) cytotoxic T lymphocytes, FOXP3(+) (Treg cell marker), and PD-1(+) immune cell counts on prognosis was analyzed utilizing immunohistochemistry. Both PD-1(+) immune cells and FOXP3(+) Tregs counts were significantly associated with unfavorable prognostic factors. In bivariate, but not multivariate analysis, high tumor infiltrating PD-1(+) cell counts correlated with significantly shorter patient survival. Our results suggest a prognostic value of the PD-1(+) immune cell population in such breast cancer patients. Targeting the PD-1 pathway may be a feasible approach to treating patients with breast cancer.

Synchronous non-small cell lung cancers: diagnostic yield can be improved by histologic and genetic methods.

PURPOSE: Current criteria for identification of synchronous non-small cell lung cancers (NSCLCs) may be confusing in patients with lymphatic metastases. This study was aimed at investigating the strategy using both the new histologic classification and driver-mutational testing to define multiple primary lung cancers. METHODS: Prospectively collected data of surgical patients with synchronous NSCLCs were retrospectively analyzed. Cases were defined using the Martini-Melamed criteria, and validated by histologic subtyping based on the new classification and driver mutation of selected genes. Survival was estimated between patients with multiple primary and metastatic disease controlling by nodal (N) stage. Factors associated with prolonged survival were evaluated using the Cox proportional hazards mode. RESULTS: A total of 131 patients followed for at least 12 months were included in this study. Controlling by N0 stage, patients who were diagnosed with multiple primary NSCLCs showed better relapse-free survival (RFS) than those with intrapulmonary metastases categorized either by the Martini-Melamed criteria or by histologic-mutational methods (both p < 0.0001). However, at N+ stage, patients stratified by Martini-Melamed criteria showed no difference in survival (p = 0.517), while those defined by histologic-mutational methods maintained superior survival compared with the control group (p = 0.042). On multivariate analysis, only N0 and diagnosis of independent lung lesions by histologic-mutational methods were significant predictors of better RFS (p = 0.031 and 0.001, respectively) CONCLUSIONS: The histologic-mutational strategy may be an option for identification of synchronous NSCLC when traditional criteria were not applicable, especially in cases with positive lymphatics. N0 stage and the diagnosis of independent pulmonary tumors were associated with better RFS.

IL-4-induced SOX9 confers lineage plasticity to aged adult lung stem cells.

Wound healing in response to acute injury is mediated by the coordinated and transient activation of parenchymal, stromal, and immune cells that resolves to homeostasis. Environmental, genetic, and epigenetic factors associated with inflammation and aging can lead to persistent activation of the microenvironment and fibrosis. Here, we identify opposing roles of interleukin-4 (IL-4) cytokine signaling in interstitial macrophages and type II alveolar epithelial cells (ATIIs). We show that IL4Ra signaling in macrophages promotes regeneration of the alveolar epithelium after bleomycin-induced lung injury. Using organoids and mouse models, we show that IL-4 directly acts on a subset of ATIIs to induce the expression of the transcription factor SOX9 and reprograms them toward a progenitor-like state with both airway and alveolar lineage potential. In the contexts of aging and bleomycin-induced lung injury, this leads to aberrant epithelial cell differentiation and bronchiolization, consistent with cellular and histological changes observed in interstitial lung disease.

Senescent cells perturb intestinal stem cell differentiation through Ptk7 induced noncanonical Wnt and YAP signaling.

Cellular senescence and the senescence-associated secretory phenotype (SASP) are implicated in aging and age-related disease, and SASP-related inflammation is thought to contribute to tissue dysfunction in aging and diseased animals. However, whether and how SASP factors influence the regenerative capacity of tissues remains unclear. Here, using intestinal organoids as a model of tissue regeneration, we show that SASP factors released by senescent fibroblasts deregulate stem cell activity and differentiation and ultimately impair crypt formation. We identify the secreted N-terminal domain of Ptk7 as a key component of the SASP that activates non-canonical Wnt / Ca2+ signaling through FZD7 in intestinal stem cells (ISCs). Changes in cytosolic [Ca2+] elicited by Ptk7 promote nuclear translocation of YAP and induce expression of YAP/TEAD target genes, impairing symmetry breaking and stem cell differentiation. Our study discovers secreted Ptk7 as a factor released by senescent cells and provides insight into the mechanism by which cellular senescence contributes to tissue dysfunction in aging and disease.

Obligate role for Rock1 and Rock2 in adult stem cell viability and function.

The ability of stem cells to rapidly proliferate and differentiate is integral to the steady-state maintenance of tissues with high turnover such as the blood and intestine. Mutations that alter these processes can cause primary immunodeficiencies, malignancies and defects in barrier function. The Rho-kinases, Rock1 and Rock2, regulate cell shape and cytoskeletal rearrangement, activities essential to mitosis. Here, we use inducible gene targeting to ablate Rock1 and Rock2 in adult mice, and identify an obligate requirement for these enzymes in the preservation of the hematopoietic and gastrointestinal systems. Hematopoietic cell progenitors devoid of Rho-kinases display cell cycle arrest, blocking the differentiation to mature blood lineages. Similarly, these mice exhibit impaired epithelial cell renewal in the small intestine, which is ultimately fatal. Our data reveal a novel role for these kinases in the proliferation and viability of stem cells and their progenitors, which is vital to maintaining the steady-state integrity of these organ systems.

Population genetics of cis-regulatory sequences that operate during embryonic development in the sea urchin Strongylocentrotus purpuratus.

Despite the fact that noncoding sequences comprise a substantial fraction of functional sites within all genomes, the evolutionary mechanisms that operate on genetic variation within regulatory elements remain poorly understood. In this study, we examine the population genetics of the core, upstream cis-regulatory regions of eight genes (AN, CyIIa, CyIIIa, Endo16, FoxB, HE, SM30 a, and SM50) that function during the early development of the purple sea urchin, Strongylocentrotus purpuratus. Quantitative and qualitative measures of segregating variation are not conspicuously different between cis-regulatory and closely linked "proxy neutral" noncoding regions containing no known functional sites. Length and compound mutations are common in noncoding sequences; conventional descriptive statistics ignore such mutations, under-representing true genetic variation by approximately 28% for these loci in this population. Patterns of variation in the cis-regulatory regions of six of the genes examined (CyIIa, CyIIIa, Endo16, FoxB, AN, and HE) are consistent with directional selection. Genetic variation within annotated transcription factor binding sites is comparable to, and frequently greater than, that of surrounding sequences. Comparisons of two paralog pairs (CyIIa/CyIIIa and AN/HE) suggest that distinct evolutionary processes have operated on their cis-regulatory regions following gene duplication. Together, these analyses provide a detailed view of the evolutionary mechanisms operating on noncoding sequences within a natural population, and underscore how little is known about how these processes operate on cis-regulatory sequences.