Noncoding genetic variation drives phenotypic diversity, but underlying mechanisms and affected cell types are incompletely understood. Here, investigation of effects of natural genetic variation on the epigenomes and transcriptomes of Kupffer cells derived from inbred mouse strains identified strain-specific environmental factors influencing Kupffer cell phenotypes, including leptin signaling in Kupffer cells from a steatohepatitis-resistant strain. Cell-autonomous and non-cell-autonomous effects of genetic variation were resolved by analysis of F1 hybrid mice and cells engrafted into an immunodeficient host. During homeostasis, non-cell-autonomous trans effects of genetic variation dominated control of Kupffer cells, while strain-specific responses to acute lipopolysaccharide injection were dominated by actions of cis-acting effects modifying response elements for lineage-determining and signal-dependent transcription factors. These findings demonstrate that epigenetic landscapes report on trans effects of genetic variation and serve as a resource for deeper analyses into genetic control of transcription in Kupffer cells and macrophages in vitro.

Microglia phenotypes are highly regulated by the brain environment, but the transcriptional networks that specify the maturation of human microglia are poorly understood. Here, we characterized stage-specific transcriptomes and epigenetic landscapes of fetal and postnatal human microglia and acquired corresponding data in induced pluripotent stem cell (iPSC)-derived microglia, in cerebral organoids, and following engraftment into humanized mice. Parallel development of computational approaches that considered transcription factor (TF) co-occurrence and enhancer activity allowed prediction of shared and state-specific gene regulatory networks associated with fetal and postnatal microglia. Additionally, many features of the human fetal-to-postnatal transition were recapitulated in a time-dependent manner following the engraftment of iPSC cells into humanized mice. These data and accompanying computational approaches will facilitate further efforts to elucidate mechanisms by which human microglia acquire stage-and disease-specific phenotypes.

ABSTRACTNoncoding genetic variation is a major driver of phenotypic diversity but determining the underlying mechanisms and the cell types in which it acts remain challenging problems. Here, we investigate the impact of natural genetic variation provided by phenotypically diverse inbred strains of mice on gene expression and epigenetic landscapes of Kupffer cells. Analysis of gene expression in Kupffer cells and other liver cell types derived from C57BL/6J, BALB/cJ and A/J mice provided evidence for strain-specific differences in environmental factors influencing Kupffer cell phenotypes, including preferential Leptin signaling in BALB/cJ Kupffer cells. Systematic analysis of transcriptomic and epigenetic data from F1 hybrids of these mice, and transcriptomic data from strain-specific Kupffer cells engrafted into a common host enabled quantitative assessment of cis versus trans effects of genetic variation on gene expression and an estimate of cell autonomous versus non cell autonomous effects. Under homeostatic conditions, trans effects of genetic variation were dominant, with the majority of trans regulation being non cell autonomous. In contrast, strain specific responses to acutely administered LPS were primarily associated with genetic variation acting in cis to modify response elements for lineage determining and signal dependent transcription factors. Collectively, these findings reveal cell intrinsic and environmental effects of natural genetic variation on gene expression, demonstrate the use of enhancers as detectors of trans effects of genetic variation, and provide a new resource for understanding the impact of genetic variation on gene expression in Kupffer cells.

Musculoskeletal modeling provides an alternative to in-vivo characteristics that are difficult to directly measure for movements such as running, especially for trunk muscles and joints. The full-body-lumbar-spine (FBLS) model by Raabe and Chaudhari, 2016 is an OpenSim model created for simulations of jogging. The lifting full-body (LFB) model by Beaucage-Gauvreau et al., 2018 is an adaptation of the FBLS created for estimating spinal loads during lifting. PURPOSE: Determine validity of the FBLS and LFB models in simulating pelvis and lumbar spine kinematics during running. METHODS: Inverse Kinematics were executed using experimental data for the FBLS and LFB models. To obtain the 3D motion data, 5 runners ran on a treadmill at self-selected jogging pace (2.6 ± 0.2 m/s). Axial rotations at the L5/S1 and the pelvis segment were calculated from marker data, and the range of motion (ROM) averaged for the experimental data and each computational model. RESULTS: FBLS and LFB models had the same relative movement patterns as the experimental data. However, the ROM for both models differed from the human data. For the experimental data, the average ROM was 33.6 ± 15.6° at the L5/S1 joint and 24.7 ± 12.3° at the pelvis. The ROM for the pelvis was 24.6 ± 12.7° and 33.1 ± 15.1° for the LFB and FBLS models, respectively. The ROM at the L5/S1 joint was much smaller than expected, at 7.3 ± 2.3° and 1.8 ± 0.5° for the LFB and FBLS models, respectively. This could be because the experimental data used a multi-segmented torso and a rigid lumbar spine marker model, whereas the OpenSim models utilized a rigid torso (lumped thoracic and cervical vertebrae, ribcage, scapulae, and head) with a coupled lumbar spine. The average maximum RMS across all participants was 0.05 ± 0.004 cm for both LFB and FBLS models. CONCLUSION: LFB model was created for lifting simulations but provides a better simulation of running motion at the lumbar and pelvis than the FBLS model, potentially due to the LFB model having a 3-DOF joint at T12/L1 and linear kinematic coupling constraints to distribute the net trunk motion across the six intervertebral joints (T12-L5). The ROM differences at the L5/S1 could potentially be corrected with a multi-segmented torso model. Both models have potential for simulating axial rotation of the pelvis and lumbar spine during running.

The fetal period is a critical time for brain development, characterized by neurogenesis, neural migration, and synaptogenesis 1-3 . Microglia, the tissue resident macrophages of the brain, are observed as early as the fourth week of gestation 4 and are thought to engage in a variety of processes essential for brain development and homeostasis 5-11 . Conversely, microglia phenotypes are highly regulated by the brain environment 12-14 . Mechanisms by which human brain development influences the maturation of microglia and microglia potential contribution to neurodevelopmental disorders remain poorly understood. Here, we performed transcriptomic analysis of human fetal and postnatal microglia and corresponding cortical tissue to define age-specific brain environmental factors that may drive microglia phenotypes. Comparative analysis of open chromatin profiles using bulk and single-cell methods in conjunction with a new computational approach that integrates epigenomic and single-cell RNA-seq data allowed decoding of cellular heterogeneity with inference of subtype- and development stage-specific transcriptional regulators. Interrogation of in vivo and in vitro iPSC-derived microglia models provides evidence for roles of putative instructive signals and downstream gene regulatory networks which establish human-specific fetal and postnatal microglia gene expression programs and potentially contribute to neurodevelopmental disorders.

Significant advancements in understanding disease mechanisms can occur through combined analysis of next-generation sequencing datasets generated using purified cell populations. Here, we detail our optimized protocol for purification of mouse hepatic macrophages (or other liver non-parenchymal populations) suitable for use in various next-generation sequencing protocols. An alternative framework is described for sorting pre-fixed hepatic nuclei populations. This strategy has the advantage of rapidly preserving the nuclei and can facilitate success with ChIP-seq for more challenging molecules.For complete details on the use and execution of these protocols, please refer to Muse et al. (2018), Sakai et al. (2019), and Seidman et al. (2020).

Kupffer cells, the resident macrophages of the liver, comprise the largest pool of tissue macrophages in the body. Within the liver sinusoids Kupffer cells perform functions common across many tissue macrophages including response to tissue damage and antigen presentation. They also engage in specialized activities including iron scavenging and the uptake of opsonized particles from the portal blood. Here, we review recent studies of the epigenetic pathways that establish Kupffer cell identity and function. We describe a model by which liver-environment specific signals induce lineage determining transcription factors necessary for differentiation of Kupffer cells from bone-marrow derived monocytes. We conclude by discussing how these lineage determining transcription factors (LDTFs) drive Kupffer cell behavior during both homeostasis and disease, with particular focus on the relevance of Kupffer cell LDTF pathways in the setting of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis.

Tissue-resident and recruited macrophages contribute to both host defense and pathology. Multiple macrophage phenotypes are represented in diseased tissues, but we lack deep understanding of mechanisms controlling diversification. Here, we investigate origins and epigenetic trajectories of hepatic macrophages during diet-induced non-alcoholic steatohepatitis (NASH). The NASH diet induced significant changes in Kupffer cell enhancers and gene expression, resulting in partial loss of Kupffer cell identity, induction of Trem2 and Cd9 expression, and cell death. Kupffer cell loss was compensated by gain of adjacent monocyte-derived macrophages that exhibited convergent epigenomes, transcriptomes, and functions. NASH-induced changes in Kupffer cell enhancers were driven by AP-1 and EGR that reprogrammed LXR functions required for Kupffer cell identity and survival to instead drive a scar-associated macrophage phenotype. These findings reveal mechanisms by which disease-associated environmental signals instruct resident and recruited macrophages to acquire distinct gene expression programs and corresponding functions.

Kupffer cells have specialized roles supporting the environment of the liver during homeostasis and disease. However, the key regulatory elements governing these behaviors are unknown. Using scRNA-seq, we found diversification of Kupffer cells and recruitment of additional macrophage subtypes during nonalcoholic steatohepatitis (NASH). A significant source of macrophage heterogeneity during NASH was traced to Cx3cr1 expressing monocytes. Further, macrophage subsets were localized in distinct niches, suggesting environmental specification as a key determinant of macrophage heterogeneity. We profiled chromatin accessibility of the major NASH associated macrophage populations to identify transcription factors governing their environmental specification. These results predict greater NFκB, RUNX, and AP1 activity in recruited hepatic macrophages compared to Kupffer cells. Surprisingly, we found minimal significant chromatin accessibility changes comparing Kupffer cells from healthy mice to mice with NASH, even though several thousand genes were differentially expressed. Instead, NASH led to altered chromatin activity, as measured by H3K27ac ChIP-seq, at Kupffer cell enhancer regions. A binding element for LXR (liver X receptor) was the top transcription factor motif identified in Kupffer cell enhancers with reduced activity during NASH. Furthermore, LXRα was required to maintain expression of a unique gene signature defining healthy Kupffer cells. Thus, our studies establish for the first time gene regulatory events controlling diverse hepatic macrophages during homeostasis and NASH.

Functional specialization of tissue resident macrophages occurs through environmental signals controlling activity and/or expression of transcription factors. Kupffer cells are resident macrophages in the hepatic sinusoids and have critical roles in the innate immune response and iron metabolism. Here, we characterize transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. Nr1h3 encoding LXRα is rapidly and highly induced in repopulating liver macrophages, suggesting its induction plays a crucial role in Kupffer cell differentiation. Restricted deletion of Nr1h3 in Kupffer cells reveal that it is required for shaping the Kupffer cell-specific enhancer landscape. Further, we obtain evidence that combinatorial interactions of DLL4 and TGF-β/BMP produced by sinusoidal endothelial cells and endogenous LXR ligands are required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of RBPJ through Notch signaling plays a key role in activating poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogram the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Using molecules which mimic these liver environment signals, we show that it is possible to induce Kupffer cell-specific genes in mouse bone marrow progenitor cells and human monocytes in vitro. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.

Tissue environment plays a powerful role in establishing and maintaining the distinct phenotypes of resident macrophages, but the underlying molecular mechanisms remain poorly understood. Here, we characterized transcriptomic and epigenetic changes in repopulating liver macrophages following acute Kupffer cell depletion as a means to infer signaling pathways and transcription factors that promote Kupffer cell differentiation. We obtained evidence that combinatorial interactions of the Notch ligand DLL4 and transforming growth factor-b (TGF-β) family ligands produced by sinusoidal endothelial cells and endogenous LXR ligands were required for the induction and maintenance of Kupffer cell identity. DLL4 regulation of the Notch transcriptional effector RBPJ activated poised enhancers to rapidly induce LXRα and other Kupffer cell lineage-determining factors. These factors in turn reprogrammed the repopulating liver macrophage enhancer landscape to converge on that of the original resident Kupffer cells. Collectively, these findings provide a framework for understanding how macrophage progenitor cells acquire tissue-specific phenotypes.

Known high-risk cutaneous malignant melanoma (CMM) genes account for melanoma risk in <40% of melanoma-prone families, suggesting the existence of additional high-risk genes or perhaps a polygenic mechanism involving multiple genetic modifiers. The goal of this study was to systematically characterize rare germline variants in 42 established melanoma genes among 144 CMM patients in 76 American CMM families without known mutations using data from whole-exome sequencing. We identified 68 rare (<0.1% in public and in-house control datasets) nonsynonymous variants in 25 genes. We technically validated all loss-of-function, inframe insertion/deletion, and missense variants predicted as deleterious, and followed them up in 1, 559 population-based CMM cases and 1, 633 controls. Several of these variants showed disease co-segregation within families. Of particular interest, a stopgain variant in TYR was present in five of six CMM cases/obligate gene carriers in one family and a single population-based CMM case. A start gain variant in the 5'UTR region of PLA2G6 and a missense variant in ATM were each seen in all three affected people in a single family, respectively. Results from rare variant burden tests showed that familial and population-based CMM patients tended to have higher frequencies of rare germline variants in albinism genes such as TYR, TYRP1, and OCA2 (P < 0.05). Our results suggest that rare nonsynonymous variants in low-or intermediate-risk CMM genes may influence familial CMM predisposition, warranting further investigation of both common and rare variants in genes affecting functionally important pathways (such as melanogenesis) in melanoma risk assessment.

Background Lung adenocarcinoma (LUAD) is the most common histologic subtype of lung cancer and has a high risk of distant metastasis at every disease stage. We aimed to characterize the genomic landscape of LUAD and identify mutation signatures associated with tumor progression. Methods and Findings We performed an integrative genomic analysis, incorporating whole exome sequencing (WES), determination of DNA copy number and DNA methylation, and transcriptome sequencing for 101 LUAD samples from the Environment And Genetics in Lung cancer Etiology (EAGLE) study. We detected driver genes by testing whether the nonsynonymous mutation rate was significantly higher than the background mutation rate and replicated our findings in public datasets with 724 samples. We performed subclonality analysis for mutations based on mutant allele data and copy number alteration data. We also tested the association between mutation signatures and clinical outcomes, including distant metastasis, survival, and tumor grade. We identified and replicated two novel candidate driver genes, POU class 4 homeobox 2 (POU4F2) (mutated in 9 [8.9%] samples) and ZKSCAN1 (mutated in 6 [5.9%] samples), and characterized their major deleterious mutations. ZKSCAN1 was part of a mutually exclusive gene set that included the RTK/RAS/RAF pathway genes BRAF, EGFR, KRAS, MET, and NF1, indicating an important driver role for this gene. Moreover, we observed strong associations between methylation in specific genomic regions and somatic mutation patterns. In the tumor evolution analysis, four driver genes had a significantly lower fraction of subclonal mutations (FSM), including TP53 (p = 0.007), KEAP1 (p = 0.012), STK11 (p = 0.0076), and EGFR (p = 0.0078), suggesting a tumor initiation role for these genes. Subclonal mutations were significantly enriched in APOBEC-related signatures (p < 2.5×10−50). The total number of somatic mutations (p = 0.0039) and the fraction of transitions (p = 5.5×10−4) were associated with increased risk of distant metastasis. Our study’s limitations include a small number of LUAD patients for subgroup analyses and a single-sample design for investigation of subclonality. Conclusions These data provide a genomic characterization of LUAD pathogenesis and progression. The distinct clonal and subclonal mutation signatures suggest possible diverse carcinogenesis pathways for endogenous and exogenous exposures, and may serve as a foundation for more effective treatments for this lethal disease. LUAD’s high heterogeneity emphasizes the need to further study this tumor type and to associate genomic findings with clinical outcomes.

PURPOSE:Despite the importance of the knee alignment, no studies have explored its effects on knee frontal plane biomechanics during cycling. The purpose of this study was to examine the effects of varus knee alignment on knee frontal plane biomechanics during stationary cycling. METHODS:Eleven subjects in each of varus and neutral groups participated in the study. Anteroposterior full limb radiographs were obtained to measure the knee mechanical axis angle, of which varus group had 174.3±1.4 degree and neutral group 179.2±1.0 degree. The subjects pedaled in six cycling conditions: 80 rpm with 0.5 kg, 1.0 kg, and 1.5 kg with and without toe cage. A nine-camera motion analysis system and an instrumented force bike pedal were utilized to collect data. A 2×2×3 (Group × Toe cage × Workload) mixed design ANOVA was used for statistical comparisons (p < 0.05). RESULTS:The peak knee abduction moment did not differ between the varus group (-7.9±1.1 Nm) and the neutral group (-6.7±1.2 Nm). The peak knee abduction angle was greater in the neutral group (-5.5±1.6°) compared to the varus group (0.02±1.6°). CONCLUSIONS:A varus knee alignment did not result in a greater peak knee abduction moment, suggesting stationary cycling may be a safe exercise prescription for people with varus knee alignment such as medial knee osteoarthritis patients.

Mounting evidence suggests that copy number variations (CNVs) can contribute to cancer susceptibility. The main goal of this study was to evaluate the role of germline CNVs in melanoma predisposition in high-risk melanoma families. We used genome-wide tiling comparative genomic hybridization and single nucleotide polymorphism arrays to characterize CNVs in 335 individuals (240 melanoma cases) from American melanoma-prone families (22 with germline CDKN2A or CDK4 mutations). We found that the global burden of overall CNVs (or deletions or duplications separately) was not significantly associated with case-control or CDKN2A/CDK4 mutation status after accounting for the familial dependence. However, we identified several rare CNVs that either involved known melanoma genes (e.g., PARP1, CDKN2A) or cosegregated with melanoma (duplication on 10q23.23, 3p12.2 and deletions on 8q424.3, 2q22.1) in families without mutations in known melanoma high-risk genes. Some of these CNVs were correlated with expression changes in disrupted genes based on RNASeq data from a subset of melanoma cases included in the CNV study. These results suggest that rare cosegregating CNVs may influence melanoma susceptibility in some melanoma-prone families and genes found in our study warrant further evaluation in future genetic analyses of melanoma.

The risk of pancreatic cancer (PC) is increased in melanoma-prone families but the causal relationship between germline CDKN2A mutations and PC risk is uncertain, suggesting the existence of non- CDKN2A factors. One genetic possibility involves patients having mutations in multiple high-risk PC-related genes; however, no systematic examination has yet been conducted. We used next-generation sequencing data to examine 24 putative PC-related genes in 43 PC patients with and 23 PC patients without germline CDKN2A mutations and 1001 controls. For each gene and the four pathways in which they occurred, we tested whether PC patients (overall or CDKN2A + and CDKN2A − cases separately) had an increased number of rare nonsynonymous variants. Overall, we identified 35 missense variants in PC patients, 14 in CDKN2A + and 21 in CDKN2A− PC cases. We found nominally significant associations for mismatch repair genes ( MLH1 , MSH2 , MSH6 , PMS2 ) in all PC patients and for ATM , CPA1 , and PMS2 in CDKN2A− PC patients. Further, nine CDKN2A + and four CDKN2A− PC patients had rare potentially deleterious variants in multiple PC-related genes. Loss-of-function variants were only observed in CDKN2A− PC patients, with ATM having the most pathogenic variants. Also, ATM variants ( n = 5) were only observed in CDKN2A− PC patients with a family history that included digestive system tumors. Our results suggest that a subset of PC patients may have increased risk because of germline mutations in multiple PC-related genes.

Ionizing radiation is an established risk factor for breast cancer. Epidemiologic studies of radiation-exposed cohorts have been primarily descriptive; molecular events responsible for the development of radiation-associated breast cancer have not been elucidated. In this study, we used array comparative genomic hybridization (array-CGH) to characterize genome-wide copy number changes in breast tumors collected in the Childhood Cancer Survivor Study (CCSS). Array-CGH data were obtained from 32 cases who developed a second primary breast cancer following chest irradiation at early ages for the treatment of their first cancers, mostly Hodgkin lymphoma. The majority of these cases developed breast cancer before age 45 (91%, n = 29), had invasive ductal tumors (81%, n = 26), estrogen receptor (ER)-positive staining (68%, n = 19 out of 28), and high proliferation as indicated by high Ki-67 staining (77%, n = 17 out of 22). Genomic regions with low-copy number gains and losses and high-level amplifications were similar to what has been reported in sporadic breast tumors, however, the frequency of amplifications of the 17q12 region containing human epidermal growth factor receptor 2 (HER2) was much higher among CCSS cases (38%, n = 12). Our findings suggest that second primary breast cancers in CCSS were enriched for an "amplifier" genomic subgroup with highly proliferative breast tumors. Future investigation in a larger irradiated cohort will be needed to confirm our findings.

Abstract Cutaneous malignant melanoma (CMM) is an etiologically heterogeneous disease with genetic, host, environmental factors, and their interactions contributing to its development. CDKN2A and CDK4 are the two established major susceptibility genes for melanoma identified so far. Recent evidence suggests that copy number variations (CNVs) may contribute to disease susceptibility in several inherited diseases including cancer. The goals of this study were 1) to assess whether the frequency of CNVs varied by CMM or CDKN2A/CDK4 mutation status; and 2) to identify rare CNVs that were related to melanoma predisposition in these high-risk families. We used genome-wide tiling CGH arrays (Nimblegen 720K exon-focused) to investigate characteristics of CNVs in 174 CMM cases, 44 high-risk unaffected family members (with dysplastic nevi/large number of moles or germline CDKN2A mutations), and 48 unrelated spouses from 50 American melanoma-prone families (21 CDKN2A+, 2 CDK4+, 27 mutation negative). We used the Nexus Copy Number™ built-in FASST2 algorithm to identify significant CNVs (significant threshold = 0.000001; minimal number of probes per segment = 5; log2 ratio>0.3 for gains and -0.3 for losses). We found that the median number of total CNVs, or gains or losses separately, did not show significant differences in CMM cases, high-risk unaffected family members, and unrelated controls. Among CMM cases, CNV frequencies were not significantly associated with germline CDKN2A/CDK4 mutation status, age at melanoma diagnosis, or number of melanomas. Similar results were obtained when number of genes and lengths of DNA segments affected by CNVs were analyzed. Restriction to large CNVs (>10 kb or >100 kb) or rare CNVs (not reported in the Toronto CNV database) did not change results significantly. On the other hand, we identified several rare large CNVs (>10 kb, not reported in unrelated controls) that either involved known melanoma genes or co-segregated with melanoma (observed in multiple CMM cases) within families. These included a 1.3Mb deletion in PARP1 in a single CMM case, a 10 kb deletion in CDKN2A in 4 of 5 CMM cases and two obligate gene carriers in a large family that was negative for CDKN2A mutations by sequencing, a 175 kb deletion in LINGO2 in 2 of 3 CMM cases in one family, a 10 kb deletion in 8q24 in 3 of 4 CMM cases in one family, a 110 kb deletion in 2q22.1 in all three cases in a family, and a 57 kb duplication in 4q32.2 in all five cases in a CDKN2A mutation positive family. The role of these CNVs, particularly those involving genes that have unknown function related to CMM development, in CMM susceptibility remain to be investigated. Citation Format: Xiaohong (Rose) Yang, Jianxin Shi, Hunter Bennett, Laura Burke, Casey Dagnall, Laurie Burdette, Belynda Hicks, Margaret Tucker, Alisa Goldstein. Germline copy number variations in melanoma families with/without CDKN2A/CDK4 mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2755. doi:10.1158/1538-7445.AM2015-2755