Lineage-specific Manner Research Articles

The Organization of the Pig T-Cell Receptor γ (TRG) Locus Provides Insights into the Evolutionary Patterns of the TRG Genes across Cetartiodactyla

The domestic pig (Sus scrofa) is a species representative of the Suina, one of the four suborders within Cetartiodactyla. In this paper, we reported our analysis of the pig TRG locus in comparison with the loci of species representative of the Ruminantia, Tylopoda, and Cetacea suborders. The pig TRG genomic structure reiterates the peculiarity of the organization of Cetartiodactyla loci in TRGC “cassettes”, each containing the basic V-J-J-C unit. Eighteen genes arranged in four TRGC cassettes, form the pig TRG locus. All the functional TRG genes were expressed, and the TRGV genes preferentially rearrange with the TRGJ genes within their own cassette, which correlates the diversity of the γ-chain repertoire with the number of cassettes. Among them, the TRGC5, located at the 5′ end of the locus, is the only cassette that retains a marked homology with the corresponding TRGC cassettes of all the analyzed species. The preservation of the TRGC5 cassette for such a long evolutionary time presumes a highly specialized function of its genes, which could be essential for the survival of species. Therefore, the maintenance of this cassette in pigs confirms that it is the most evolutionarily ancient within Cetartiodactyla, and it has undergone a process of duplication to give rise to the other TRGC cassettes in the different artiodactyl species in a lineage-specific manner.

Phenolic degradation by catechol dioxygenases is associated with pathogenic fungi with a necrotrophic lifestyle in the Ceratocystidaceae.

Fungal species of the Ceratocystidaceae grow on their host plants using a variety of different lifestyles, from saprophytic to highly pathogenic. Although many genomes of fungi in the Ceratocystidaceae are publicly available, it is not known how the genes that encode catechol dioxygenases (CDOs), enzymes involved in the degradation of phenolic plant defense compounds, differ among members of the Ceratocystidaceae. The aim of this study was therefore to identify and characterize the genes encoding CDOs in the genomes of Ceratocystidaceae representatives. We found that genes encoding CDOs are more abundant in pathogenic necrotrophic species of the Ceratocystidaceae and less abundant in saprophytic species. The loss of the CDO genes and the associated 3-oxoadipate catabolic pathway appears to have occurred in a lineage-specific manner. Taken together, this study revealed a positive association between CDO gene copy number and fungal lifestyle in Ceratocystidaceae representatives.

The Downregulation of Both Giant HERCs, HERC1 and HERC2, Is an Unambiguous Feature of Chronic Myeloid Leukemia, and HERC1 Levels Are Associated with Leukemic Cell Differentiation.

Large HERC E3 ubiquitin ligase family members, HERC1 and HERC2, are staggeringly complex proteins that can intervene in a wide range of biological processes, such as cell proliferation, DNA repair, neurodevelopment, and inflammation. Therefore, mutations or dysregulation of large HERCs is associated with neurological disorders, DNA repair defects, and cancer. Though their role in solid tumors started to be investigated some years ago, our knowledge about HERCs in non-solid neoplasm is greatly lagging behind. Chronic Myeloid Leukemia (CML) is a model onco-hematological disorder because of its unique and unambiguous relation between genotype and phenotype due to a single genetic alteration. In the present study, we ascertained that the presence of the BCR-ABL fusion gene was inversely associated with the expression of the HERC1 and HERC2 genes. Upon the achievement of remission, both HERC1 and HERC2 mRNAs raised again to levels comparable to those of the healthy donors. Additionally, our survey unveiled that their gene expression is sensitive to different Tyrosine Kinases Inhibitors (TKIs) in a time-dependent fashion. Interestingly, for the first time, we also observed a differential HERC1 expression when the leukemic cell lines were induced to differentiate towards different lineages revealing that HERC1 protein expression is associated with the differentiation process in a lineage-specific manner. Taken together, our findings suggest that HERC1 might act as a novel potential player in blood cell differentiation. Overall, we believe that our results are beneficial to initiate exploring the role/s of large HERCs in non-solid neoplasms.

A prelude to the proximity interaction mapping of CXXC5

CXXC5 is a member of the zinc-finger CXXC family proteins that interact with unmodified CpG dinucleotides through a conserved ZF-CXXC domain. CXXC5 is involved in the modulation of gene expressions that lead to alterations in diverse cellular events. However, the underlying mechanism of CXXC5-modulated gene expressions remains unclear. Proteins perform their functions in a network of proteins whose identities and amounts change spatiotemporally in response to various stimuli in a lineage-specific manner. Since CXXC5 lacks an intrinsic transcription regulatory function or enzymatic activity but is a DNA binder, CXXC5 by interacting with proteins could act as a scaffold to establish a chromatin state restrictive or permissive for transcription. To initially address this, we utilized the proximity-dependent biotinylation approach. Proximity interaction partners of CXXC5 include DNA and chromatin modifiers, transcription factors/co-regulators, and RNA processors. Of these, CXXC5 through its CXXC domain interacted with EMD, MAZ, and MeCP2. Furthermore, an interplay between CXXC5 and MeCP2 was critical for a subset of CXXC5 target gene expressions. It appears that CXXC5 may act as a nucleation factor in modulating gene expressions. Providing a prelude for CXXC5 actions, our results could also contribute to a better understanding of CXXC5-mediated cellular processes in physiology and pathophysiology.

Evolutionary conservation of human ketodeoxynonulosonic acid production is independent of sialoglycan biosynthesis.

Human metabolic incorporation of nonhuman sialic acid (Sia) N-glycolylneuraminic acid into endogenous glycans generates inflammation via preexisting antibodies, which likely contributes to red meat-induced atherosclerosis acceleration. Exploring whether this mechanism affects atherosclerosis in end-stage renal disease (ESRD), we instead found serum accumulation of 2-keto-3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (Kdn), a Sia prominently expressed in cold-blooded vertebrates. In patients with ESRD, levels of the Kdn precursor mannose also increased, but within a normal range. Mannose ingestion by healthy volunteers raised the levels of urinary mannose and Kdn. Kdn production pathways remained conserved in mammals but were diminished by an M42T substitution in a key biosynthetic enzyme, N-acetylneuraminate synthase. Remarkably, reversion to the ancestral methionine then occurred independently in 2 lineages, including humans. However, mammalian glycan databases contain no Kdn-glycans. We hypothesize that the potential toxicity of excess mannose in mammals is partly buffered by conversion to free Kdn. Thus, mammals probably conserve Kdn biosynthesis and modulate it in a lineage-specific manner, not for glycosylation, but to control physiological mannose intermediates and metabolites. However, human cells can be forced to express Kdn-glycans via genetic mutations enhancing Kdn utilization, or by transfection with fish enzymes producing cytidine monophosphate-Kdn (CMP-Kdn). Antibodies against Kdn-glycans occur in pooled human immunoglobulins. Pathological conditions that elevate Kdn levels could therefore result in antibody-mediated inflammatory pathologies.

Ddx41 loss R-loops in cGAS to fuel inflammatory HSPC production

In this issue of Developmental Cell, Weinreb etal. reveal that loss of the DEAD-box helicase Ddx41 unexpectedly triggers an R-loop-mediated sterile inflammatory cascade which drives HSPC production during embryonic development. Human studies suggest mechanistic conservation for inflammation in DDX41-associated hematologic disease, uncovering a potential route for future therapeutic intervention.

Structural phylogenetic analysis reveals lineage-specific RNA repetitive structural motifs in all coronaviruses and associated variations in SARS-CoV-2.

In many single-stranded (ss) RNA viruses, the cis-acting packaging signal that confers selectivity genome packaging usually encompasses short structured RNA repeats. These structural units, termed repetitive structural motifs (RSMs), potentially mediate capsid assembly by specific RNA–protein interactions. However, general knowledge of the conservation and/or the diversity of RSMs in the positive-sense ssRNA coronaviruses (CoVs) is limited. By performing structural phylogenetic analysis, we identified a variety of RSMs in nearly all CoV genomic RNAs, which are exclusively located in the 5′-untranslated regions (UTRs) and/or in the inter-domain regions of poly-protein 1ab coding sequences in a lineage-specific manner. In all alpha- and beta-CoVs, except for Embecovirus spp, two to four copies of 5′-gUUYCGUc-3′ RSMs displaying conserved hexa-loop sequences were generally identified in Stem-loop 5 (SL5) located in the 5′-UTRs of genomic RNAs. In Embecovirus spp., however, two to eight copies of 5′-agc-3′/guAAu RSMs were found in the coding regions of non-structural protein (NSP) 3 and/or NSP15 in open reading frame (ORF) 1ab. In gamma- and delta-CoVs, other types of RSMs were found in several clustered structural elements in 5′-UTRs and/or ORF1ab. The identification of RSM-encompassing structural elements in all CoVs suggests that these RNA elements play fundamental roles in the life cycle of CoVs. In the recently emerged SARS-CoV-2, beta-CoV-specific RSMs are also found in its SL5, displaying two copies of 5′-gUUUCGUc-3′ motifs. However, multiple sequence alignment reveals that the majority of SARS-CoV-2 possesses a variant RSM harboring SL5b C241U, and intriguingly, several variations in the coding sequences of viral proteins, such as Nsp12 P323L, S protein D614G, and N protein R203K-G204R, are concurrently found with such variant RSM. In conclusion, the comprehensive exploration for RSMs reveals phylogenetic insights into the RNA structural elements in CoVs as a whole and provides a new perspective on variations currently found in SARS-CoV-2.

Myeloid-Derived Suppressor Cell Differentiation in Cancer: Transcriptional Regulators and Enhanceosome-Mediated Mechanisms.

Myeloid-derived Suppressor Cells (MDSCs) are a sub-population of leukocytes that are important for carcinogenesis and cancer immunotherapy. During carcinogenesis or severe infections, inflammatory mediators induce MDSCs via aberrant differentiation of myeloid precursors. Although several transcription factors, including C/EBPβ, STAT3, c-Rel, STAT5, and IRF8, have been reported to regulate MDSC differentiation, none of them are specifically expressed in MDSCs. How these lineage-non-specific transcription factors specify MDSC differentiation in a lineage-specific manner is unclear. The recent discovery of the c-Rel−C/EBPβ enhanceosome in MDSCs may help explain these context-dependent roles. In this review, we examine several transcriptional regulators of MDSC differentiation, and discuss the concept of non-modular regulation of MDSC signature gene expression by transcription factors such as c-Rel and C/EBPß.

An in vivo screen of noncoding loci reveals that Daedalus is a gatekeeper of an Ikaros-dependent checkpoint during haematopoiesis

Haematopoiesis relies on tightly controlled gene expression patterns as development proceeds through a series of progenitors. While the regulation of hematopoietic development has been well studied, the role of noncoding elements in this critical process is a developing field. In particular, the discovery of new regulators of lymphopoiesis could have important implications for our understanding of the adaptive immune system and disease. Here we elucidate how a noncoding element is capable of regulating a broadly expressed transcription factor, Ikaros, in a lymphoid lineage-specific manner, such that it imbues Ikaros with the ability to specify the lymphoid lineage over alternate fates. Deletion of the Daedalus locus, which is proximal to Ikaros, led to a severe reduction in early lymphoid progenitors, exerting control over the earliest fate decisions during lymphoid lineage commitment. Daedalus locus deletion led to alterations in Ikaros isoform expression and a significant reduction in Ikaros protein. The Daedalus locus may function through direct DNA interaction as Hi-C analysis demonstrated an interaction between the two loci. Finally, we identify an Ikaros-regulated erythroid-lymphoid checkpoint that is governed by Daedalus in a lymphoid-lineage-specific manner. Daedalus appears to act as a gatekeeper of Ikaros's broad lineage-specifying functions, selectively stabilizing Ikaros activity in the lymphoid lineage and permitting diversion to the erythroid fate in its absence. These findings represent a key illustration of how a transcription factor with broad lineage expression must work in concert with noncoding elements to orchestrate hematopoietic lineage commitment.

STAT5 promotes accessibility and is required for BATF-mediated plasticity at the Il9 locus

T helper cell differentiation requires lineage-defining transcription factors and factors that have shared expression among multiple subsets. BATF is required for development of multiple Th subsets but functions in a lineage-specific manner. BATF is required for IL-9 production in Th9 cells but in contrast to its function as a pioneer factor in Th17 cells, BATF is neither sufficient nor required for accessibility at the Il9 locus. Here we show that STAT5 is the earliest factor binding and remodeling the Il9 locus to allow BATF binding in both mouse and human Th9 cultures. The ability of STAT5 to mediate accessibility for BATF is observed in other Th lineages and allows acquisition of the IL-9-secreting phenotype. STAT5 and BATF convert Th17 cells into cells that mediate IL-9-dependent effects in allergic airway inflammation and anti-tumor immunity. Thus, BATF requires the STAT5 signal to mediate plasticity at the Il9 locus.

Evolution of SHORT VEGETATIVE PHASE (SVP) genes in Rosaceae: Implications of lineage-specific gene duplication events and function diversifications with respect to their roles in processes other than bud dormancy.

MADS-box genes that are homologous to Arabidopsis SHORT VEGETATIVE PHASE (SVP) have been shown to play key roles in the regulation of bud dormancy in perennial species, particularly in the deciduous fruit trees of Rosaceae. However, their evolutionary profiles in Rosaceae have not yet been analyzed systematically. Here, The SVP genes were found to be significantly expanded in Rosaceae when compared with annual species from Brassicaceae. Phylogenetic analysis showed that Rosaceae SVP genes could be classified into five clades, namely, SVP1, SVP2-R1, SVP2-R2, SVP2-R3 and SVP3. The SVP1 clade genes were retained in most of the species, whereas the SVP2-R2 and SVP2-R3 clades were found to be Maleae- and Amygdaleae-specific (Both of the lineages belong to Amygdaloideae), respectively, and SVP2-R1 was Rosoideae-specific in Rosaceae. Furthermore, 10 lineage-specific gene duplication (GD) events (GD1-10) were proposed for the expansion of SVP genes, suggesting that the expansion and divergence of Rosaceae SVP genes were mainly derived by lineage-specific manner during evolution. Moreover, tandem and segmental duplications were the major reasons for the expansion of SVP genes, and interestingly, tandem duplications, a well-known evolutionary feature of SVP genes, were found to be mainly Amygdaloideae-specific. Sequence alignment, selection pressure, and cis-acting element analysis suggested large functional innovations and diversification of SVP genes in different lineages of Rosaceae. Finally, the different growth cycle of Rosa multiflora and their novel expression patterns of RmSVP genes provided new insights into the functional diversification of SVP genes in terms of their roles in processes other than bud dormancy.

Lineage-Specific Epigenomic and Genomic Activation of Oncogene HNF4A Promotes Gastrointestinal Adenocarcinomas.

Gastrointestinal adenocarcinomas (GIAC) of the tubular gastrointestinal (GI) tract including esophagus, stomach, colon, and rectum comprise most GI cancers and share a spectrum of genomic features. However, the unified epigenomic changes specific to GIAC are poorly characterized. Using 907 GIAC samples from The Cancer Genome Atlas, we applied mathematical algorithms to large-scale DNA methylome and transcriptome profiles to reconstruct transcription factor (TF) networks and identify a list of functionally hyperactive master regulator (MR) TF shared across different GIAC. The top candidate HNF4A exhibited prominent genomic and epigenomic activation in a GIAC-specific manner. A complex interplay between the HNF4A promoter and three distal enhancer elements was coordinated by GIAC-specific MRTF including ELF3, GATA4, GATA6, and KLF5. HNF4A also self-regulated its own promoter and enhancers. Functionally, HNF4A promoted cancer proliferation and survival by transcriptional activation of many downstream targets, including HNF1A and factors of interleukin signaling, in a lineage-specific manner. Overall, our study provides new insights into the GIAC-specific gene regulatory networks and identifies potential therapeutic strategies against these common cancers. SIGNIFICANCE: These findings show that GIAC-specific master regulatory transcription factors control HNF4A via three distal enhancers to promote GIAC cell proliferation and survival. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/13/2722/F1.large.jpg.

The Complete Plastomes of Five Hemiparasitic Plants (Osyris wightiana, Pyrularia edulis, Santalum album, Viscum liquidambaricolum, and V. ovalifolium): Comparative and Evolutionary Analyses Within Santalales.

Most species of Santalales (the sandalwood order) are hemiparasites, including both facultative and obligate hemiparasites. Despite its rich diversity, only a small fraction of the species in the sandalwood order have sequenced plastomes. The evolution of parasitism–associated plastome reduction in Santalales remains under-studied. Here, we report the complete plastomes of three facultative hemiparasites (Pyrularia edulis, Cervantesiaceae; Osyris wightiana, and Santalum album, Santalaceae), and two obligate hemiparasites (Viscum liquidambaricolum and Viscum ovalifolium, Viscaceae). Coupled with publicly available data, we investigated the dynamics of plastome degradation in Santalales hemiparasites. Our results indicate that these hemiparasites can be characterized by various degrees of plastome downsizing, structural rearrangement, and gene loss. The loss or pseudogenization of ndh genes was commonly observed in Santalales hemiparasites, which may be correlated to the lifestyle shift from photoautotroph to hemiparasitism. However, the obligate hemiparasites did not exhibit a consistently higher level of gene loss or pseudogenization compared to facultative hemiparasites, which suggests that the degree of plastome reduction is not correlated with the trophic level facultative or obligate hemiparasitism. Instead, closely related taxa tend to possess highly similar plastome size, structure, and gene content. This implies the parasitism-associated plastome degradation in Santalales may evolve in a lineage-specific manner.

Comprehensive analysis of Translationally Controlled Tumor Protein (TCTP) provides insights for lineage-specific evolution and functional divergence.

BackgroundTranslationally controlled tumor protein (TCTP) is a conserved, multifunctional protein involved in numerous cellular processes in eukaryotes. Although the functions of TCTP have been investigated sporadically in animals, invertebrates, and plants, few lineage-specific activities of this molecule, have been reported. An exception is in Arabidopsis thaliana, in which TCTP (AtTCTP1) functions in stomatal closuer by regulating microtubule stability. Further, although the development of next-generation sequencing technologies has facilitated the analysis of many eukaryotic genomes in public databases, inter-kingdom comparative analyses using available genome information are comparatively scarce.MethodologyTo carry out inter-kingdom comparative analysis of TCTP, TCTP genes were identified from 377 species. Then phylogenetic analysis, prediction of protein structure, molecular docking simulation and molecular dynamics analysis were performed to investigate the evolution of TCTP genes and their binding proteins.ResultsA total of 533 TCTP genes were identified from 377 eukaryotic species, including protozoa, fungi, invertebrates, vertebrates, and plants. Phylogenetic and secondary structure analyses reveal lineage-specific evolution of TCTP, and inter-kingdom comparisons highlight the lineage-specific emergence of, or changes in, secondary structure elements in TCTP proteins from different kingdoms. Furthermore, secondary structure comparisons between TCTP proteins within each kingdom, combined with measurements of the degree of sequence conservation, suggest that TCTP genes have evolved to conserve protein secondary structures in a lineage-specific manner. Additional tertiary structure analysis of TCTP-binding proteins and their interacting partners and docking simulations between these proteins further imply that TCTP gene variation may influence the tertiary structures of TCTP-binding proteins in a lineage-specific manner.ConclusionsOur analysis suggests that TCTP has undergone lineage-specific evolution and that structural changes in TCTP proteins may correlate with the tertiary structure of TCTP-binding proteins and their binding partners in a lineage-specific manner.

H3K27me3 demethylase UTX regulates the differentiation of a subset of bipolar cells in the mouse retina.

Di- and trimethylation of lysine 27 on histone 3 (H3K27me2/3) is a critical gene repression mechanism. We previously showed that down-regulation of the H3K27 demethylase, Jumonji domain-containing protein 3 (JMJD3), resulted in a reduced number of protein kinase C (PKC)α-positive rod ON-bipolar cells. In this work, we focused on the role of another H3K27 demethylase, ubiquitously transcribed tetratricopeptide repeat X chromosome (UTX), in retinal development. UTX was expressed in the retinal progenitor cells of the embryonic mouse retina and was observed in the inner nuclear layer during late retinal development and in the mature retina. The short hairpin RNA-mediated knockdown of Utx in a mouse retinal explant led to a reduced number of PKCα-positive rod ON-bipolar cells. However, other retinal subtypes were unaffected by this knockdown. Using a retina-specific knockout of Utx in mice, the in vivo effects of UTX down-regulation were examined. Again, the number of PKCα-positive rod ON-bipolar cells was reduced, and no other apparent phenotypes, including retinal progenitor proliferation, apoptosis or differentiation, were observed. Finally, we examined retina-specific Utx and Jmjd3 double-knockout mice and found that although the number of rod ON-bipolar cells was reduced, no additional effects from the loss of Utx and Jmjd3 were observed. Taken together, our data show that UTX contributes to retinal differentiation in a lineage-specific manner.

Metallomics reveals a persisting impact of cadmium on the evolution of metal-selective snail metallothioneins.

The tiny contribution of cadmium (Cd) to the composition of the earth's crust contrasts with its high biological significance, owing mainly to the competition of Cd with the essential zinc (Zn) for suitable metal binding sites in proteins. In this context it was speculated that in several animal lineages, the protein family of metallothioneins (MTs) has evolved to specifically detoxify Cd. Although the multi-functionality and heterometallic composition of MTs in most animal species does not support such an assumption, there are some exceptions to this role, particularly in animal lineages at the roots of animal evolution. In order to substantiate this hypothesis and to further understand MT evolution, we have studied MTs of different snails that exhibit clear Cd-binding preferences in a lineage-specific manner. By applying a metallomics approach including 74 MT sequences from 47 gastropod species, and by combining phylogenomic methods with molecular, biochemical, and spectroscopic techniques, we show that Cd selectivity of snail MTs has resulted from convergent evolution of metal-binding domains that significantly differ in their primary structure. We also demonstrate how their Cd selectivity and specificity has been optimized by the persistent impact of Cd through 430 million years of MT evolution, modifying them upon lineage-specific adaptation of snails to different habitats. Overall, our results support the role of Cd for MT evolution in snails, and provide an interesting example of a vestigial abiotic factor directly driving gene evolution. Finally, we discuss the potential implications of our findings for studies devoted to the understanding of mechanisms leading to metal specificity in proteins, which is important when designing metal-selective peptides.

Abstract P4-04-04: Loss of epigenetic lineage fidelity with age primes breast epithelia for malignant transformation

Abstract Background: Aging is the largest risk factor for breast cancer, with 80% of new cases diagnosed in women over 50y. However, the molecular mechanism underlying age-associated cancer susceptibility is still not well understood. While aging-associated phenotypes in a number of tissues have been correlated with transcriptional and epigenetic changes, few studies have looked at epigenetic regulation at the resolution of individual cell populations. The bilayered epithelia of the mammary gland is composed of an apical layer of secretory luminal epithelial cells (LEP) surrounded by a basal layer of contractile and tumor suppressive myoepithelial cells (MEP). Breast cancer cells of origin are thought to reside mainly in the luminal or supra-basal regions directly adjacent to the apical surfaces of the MEP. Thus, an understanding of how LEP and MEP interact and maintain lineage-specific expression, and how this is dysregulated with age may elucidate a potential mechanism regulating increased breast cancer susceptibility with age. Previously, we showed that loss of lineage fidelity of LEP and MEP is a key feature of aging. Moreover, this loss of lineage fidelity can be imposed by old MEP onto young LEP in a cell non-autonomous manner (Miyano & Sayaman, et al, Aging 2017). Methods: We present a detailed analysis of genome-wide DNA methylation (Infinium Human Methylation 450K) changes at lineage-specific resolution from FACS-sorted MEP and LEP populations isolated from finite lifespan human mammary epithelial cells (HMEC) derived from reduction mammoplasties of younger (<30y) and older women (>55y). Results: In younger epithelia, maintenance of lineage fidelity is associated with lineage-specific differential methylation (DM) of CpG sites at key regulatory regions. We find that loss of lineage fidelity is recapitulated in genome-wide DNA methylation, with loss of DM at canonical proximal regions associated with transcriptional regulation, specifically annotated CpG island groups, enhancer regions, and CTCF binding sites thought to be involved in chromatin remodeling. Strikingly, DM changes with age occur almost exclusively in LEP population, with older LEP acquiring more MEP-like DNA methylation patterns at the dysregulated sites. Aged LEP and MEP also exhibit significant variability in DNA methylation at both hypo- and hypermethylated regions that show little variance in young samples. Furthermore, predictions of physiologic age using Horvath’s clock show MEP to have older physiologic ages than their isogenic LEP counterparts. Titus et al, found that DM regions (DMRs) between early stage breast tumors and normal-adjacent breast samples in The Cancer Genome Atlas (TCGA) are significantly more enriched in the LUMA, LUMB and HER2 than the Basal molecular subtypes (Sci Reports, 2017). We show that CpG sites associated with the luminal-subtype DMRs are lineage specific and that a significant fraction of the epigenetic changes that occur in these early stage breast cancers are already present during aging. Conclusions: The lineage bias of age-specific of DNA methylation changes suggests that epithelial lineages age via different mechanisms. That LEP are significantly more differentially regulated via DNA methylation with age than MEP raises possibilities that DNA methylation affects aging-associated breast cancer risk in a lineage-specific manner. This lineage bias, along with age-specific changes in breast composition, may underlie the differences in incidence rates of breast cancer subtypes with age and the prevalence of ER+ luminal-subtype breast cancers in women post-menopause. We suggest that these epigenetic changes in the luminal lineage may be priming events that make breast cells more vulnerable to further dysregulation that leads to cancer. Citation Format: Rosalyn W. Sayaman, Masaru Miyano, Mark A. LaBarge. Loss of epigenetic lineage fidelity with age primes breast epithelia for malignant transformation [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-04-04.

Cadmium Pathways in Snails Follow a Complementary Strategy between Metallothionein Detoxification and Auxiliary Inactivation by Phytochelatins.

Metal detoxification is crucial for animals to cope with environmental exposure. In snails, a pivotal role in protection against cadmium (Cd) is attributed to metallothioneins (MTs). Some gastropod species express, in a lineage-specific manner, Cd-selective MTs devoted exclusively to the binding and detoxification of this single metal, whereas other species of snails possess non-selective MTs, but still show a high tolerance against Cd. An explanation for this may be that invertebrates and in particular snails may also synthetize phytochelatins (PCs), originally known to be produced by plants, to provide protection against metal or metalloid toxicity. Here we demonstrate that despite the fact that similar mechanisms for Cd inactivation exist in snail species through binding of the metal to MTs, the actual detoxification pathways for this metal may follow different traits in a species-specific manner. In particular, this depends on the detoxification capacity of MTs due to their Cd-selective or non-specific binding features. In the terrestrial slug Arion vulgaris, for example, Cd is solely detoxified by a Cd-selective MT isoform (AvMT1). In contrast, the freshwater snail Biomphalaria glabrata activates an additional pathway for metal inactivation by synthesizing phytochelatins, which compensate for the insufficient capacity of its non-selective MT system to detoxify Cd. We hypothesize that in other snails and invertebrate species, too, an alternative inactivation of the metal by PCs may occur, if their MT system is not Cd-selective enough, or its Cd loading capacity is exhausted.

Artery-Associated Sympathetic Innervation Drives Rhythmic Vascular Inflammation of Arteries and Veins.

The incidence of acute cardiovascular complications is highly time-of-day dependent. However, the mechanisms driving rhythmicity of ischemic vascular events are unknown. Although enhanced numbers of leukocytes have been linked to an increased risk of cardiovascular complications, the role that rhythmic leukocyte adhesion plays in different vascular beds has not been studied. We evaluated leukocyte recruitment in vivo by using real-time multichannel fluorescence intravital microscopy of a tumor necrosis factor-α-induced acute inflammation model in both murine arterial and venous macrovasculature and microvasculature. These approaches were complemented with genetic, surgical, and pharmacological ablation of sympathetic nerves or adrenergic receptors to assess their relevance for rhythmic leukocyte adhesion. In addition, we genetically targeted the key circadian clock gene Bmal1 (also known as Arntl) in a lineage-specific manner to dissect the importance of oscillations in leukocytes and components of the vessel wall in this process. In vivo quantitative imaging analyses of acute inflammation revealed a 24-hour rhythm in leukocyte recruitment to arteries and veins of the mouse macrovasculature and microvasculature. Unexpectedly, although in arteries leukocyte adhesion was highest in the morning, it peaked at night in veins. This phase shift was governed by a rhythmic microenvironment and a vessel type-specific oscillatory pattern in the expression of promigratory molecules. Differences in cell adhesion molecules and leukocyte adhesion were ablated when disrupting sympathetic nerves, demonstrating their critical role in this process and the importance of β2-adrenergic receptor signaling. Loss of the core clock gene Bmal1 in leukocytes, endothelial cells, or arterial mural cells affected the oscillations in a vessel type-specific manner. Rhythmicity in the intravascular reactivity of adherent leukocytes resulted in increased interactions with platelets in the morning in arteries and in veins at night with a higher predisposition to acute thrombosis at different times as a consequence. Together, our findings point to an important and previously unrecognized role of artery-associated sympathetic innervation in governing rhythmicity in vascular inflammation in both arteries and veins and its potential implications in the occurrence of time-of-day-dependent vessel type-specific thrombotic events.

Transcriptional Enhancers in the FOXP2 Locus Underwent Accelerated Evolution in the Human Lineage.

Unique human features, such as complex language, are the result of molecular evolutionary changes that modified developmental programs of our brain. The human-specific evolution of the forkhead box P2 (FOXP2) gene-coding region has been linked to the emergence of speech and language in the human kind. However, little is known about how the expression of FOXP2 is regulated and whether its regulatory machinery evolved in a lineage-specific manner in humans. In order to identify FOXP2 regulatory regions containing human-specific changes, we used databases of human-accelerated noncoding sequences or HARs. We found that the topologically associating domain determined using developing human cerebral cortex containing the FOXP2 locus includes two clusters of 12 HARs, placing the locus occupied by FOXP2 among the top regions showing fast acceleration rates in noncoding regions in the human genome. Using in vivo enhancer assays in zebrafish, we found that at least five FOXP2-HARs behave as transcriptional enhancers throughout different developmental stages. In addition, we found that at least two FOXP2-HARs direct the expression of the reporter gene EGFP to foxP2-expressing regions and cells. Moreover, we uncovered two FOXP2-HARs showing reporter expression gain of function in the nervous system when compared with the chimpanzee ortholog sequences. Our results indicate that regulatory sequences in the FOXP2 locus underwent a human-specific evolutionary process suggesting that the transcriptional machinery controlling this gene could have also evolved differentially in the human lineage.