The Landscape of L1 Retrotransposons in the Human Genome Is Shaped by Pre-insertion Sequence Biases and Post-insertion Selection.

L1 retrotransposons are transposable elements and major contributors of genetic variation in humans. Where L1 integrates into the genome can directly impact human evolution and disease. Here, we experimentally induced L1 retrotransposition in cells and mapped integration sites at nucleotide resolution. At local scales, L1 integration is mostly restricted by genome sequence biases and the specificity of the L1 machinery. At regional scales, L1 shows a broad capacity for integration into all chromatin states, in contrast to other known mobile genetic elements. However, integration is influenced by the replication timing of target regions, suggesting a link to host DNA replication. The distribution of new L1 integrations differs from those of preexisting L1 copies, which are significantly reshaped by natural selection. Our findings reveal that the L1 machinery has evolved to efficiently target all genomic regions and underline a predominant role for post-integrative processes on the distribution of endogenous L1 elements.

Short tandem repeats are important contributors to silencer elements in T cells.

The action of cis-regulatory elements with either activation or repression functions underpins the precise regulation of gene expression during normal development and cell differentiation. Gene activation by the combined activities of promoters and distal enhancers has been extensively studied in normal and pathological contexts. In sharp contrast, gene repression by cis-acting silencers, defined as genetic elements that negatively regulate gene transcription in a position-independent fashion, is less well understood. Here, we repurpose the STARR-seq approach as a novel high-throughput reporter strategy to quantitatively assess silencer activity in mammals. We assessed silencer activity from DNase hypersensitive I sites in a mouse T cell line. Identified silencers were associated with either repressive or active chromatin marks and enriched for binding motifs of known transcriptional repressors. CRISPR-mediated genomic deletions validated the repressive function of distinct silencers involved in the repression of non-T cell genes and genes regulated during T cell differentiation. Finally, we unravel an association of silencer activity with short tandem repeats, highlighting the role of repetitive elements in silencer activity. Our results provide a general strategy for genome-wide identification and characterization of silencer elements.

Zbtb7a is a transducer for the control of promoter accessibility by NF-kappa B and multiple other transcription factors.

Gene expression in eukaryotes is controlled by DNA sequences at promoter and enhancer regions, whose accessibility for binding by regulatory proteins dictates their specific patterns of activity. Here, we identify the protein Zbtb7a as a factor required for inducible changes in accessibility driven by transcription factors (TFs). We show that Zbtb7a binds to a significant fraction of genomic promoters and enhancers, encompassing many target genes of nuclear factor kappa B (NFκB) p65 and a variety of other TFs. While Zbtb7a binding is not alone sufficient to directly activate promoters, it is required to enable TF-dependent control of accessibility and normal gene expression. Using p65 as a model TF, we show that Zbtb7a associates with promoters independently of client TF binding. Moreover, the presence of prebound Zbtb7a can specify promoters that are amenable to TF-induced changes in accessibility. Therefore, Zbtb7a represents a widely used promoter factor that transduces signals from other TFs to enable control of accessibility and regulation of gene expression.

Cell-type-specific control of enhancer activity by H3K9 trimethylation.

Cell-type-specific control of gene expression is critical for the development of multicellular organisms. To investigate the mechanisms which underlie this, we have studied the regulation of the model genes Mdc and Il12b, whose stimulus-induced expression is tightly restricted to specific cells of the immune system. Surprisingly, we find that neither the promoter nor the enhancer sequences of these genes are sufficient to direct this cell-type specificity. Instead, the activities of upstream enhancers are repressed in nonexpressing cells by high levels of trimethylated H3K9 in their flanking regions. Genome-wide analysis indicates that this manner of regulation is shared by numerous enhancers of cell-type-specific genes. In dendritic cells and macrophages, the stimulus-induced demethylase Jmjd2d controls H3K9me3 levels at these regions, and is thereby required for Mdc and Il12b transcription. By experimentally assaying multiple enhancers in a variety of cell types, we show that regulation by H3K9me3 is a widely used mechanism which imparts specificity to the activities of otherwise broadly functional enhancers.

Regulation of stimulus-inducible gene expression in myeloid cells.

One of the best-characterized and biologically important gene expression programmes in myeloid cells is their response to pro-inflammatory stimuli. Macrophages and DCs in particular are key mediators of immune responses, and are widely-used as prototypes to understand and define the determinants of specific and inducible gene expression. In this review we summarize advances and concepts which have been made towards the understanding of inducible gene expression, with a particular focus on insights gained using the myeloid system as a model. We discuss the emerging concept of layered control of gene regulation and cell identity by different functional classes of transcription factors; and examine recent progress to understanding the molecular processes involved, including the involvement of nucleosome positioning, chromatin modifications, and nuclear architecture. We also address the exciting but less-well understood role of non-coding RNAs in controlling specific gene expression programmes in myeloid and other cell-types.

A feed-forward circuit controlling inducible NF-κB target gene activation by promoter histone demethylation.

Activation of transcription from a silenced state is crucial to achieve specific gene expression in many biological contexts. Methylation of lysine 9 on histone H3 (H3K9) is widely associated with transcriptional silencing, and its disappearance is linked to the activation of several inflammatory genes by NF-κB. Here we describe that this event is controlled by a feed-forward circuit catalyzed by the activity of the histone demethylase Aof1 (also known as Lsd2/Kdm1b). We find that Aof1 is required for removal of dimethyl H3K9 at specific promoters, and thereby it controls stimulus-induced recruitment of NF-κB and gene expression. However, Aof1 is itself recruited by interaction with the c-Rel subunit of NF-κB, which is found at low levels associated with promoters in unstimulated cells. Thus, at these tightly regulated genes, NF-κB functions both as a transcriptional activator and as an upstream targeting signal that marks promoters to be derepressed by histone demethylation.

Locus-level L1 DNA methylation profiling reveals the epigenetic and transcriptional interplay between L1s and their integration sites.

Long interspersed element 1 (L1) retrotransposons are implicated in human disease and evolution. Their global activity is repressed by DNA methylation, but deciphering the regulation of individual copies has been challenging. Here, we combine short- and long-read sequencing to unveil L1 methylation heterogeneity across cell types, families, and individual loci and elucidate key principles involved. We find that the youngest primate L1 families are specifically hypomethylated in pluripotent stem cells and the placenta but not in most tumors. Locally, intronic L1 methylation is intimately associated with gene transcription. Conversely, the L1 methylation state can propagate to the proximal region up to 300 bp. This phenomenon is accompanied by the binding of specific transcription factors, which drive the expression of L1 and chimeric transcripts. Finally, L1 hypomethylation alone is typically insufficient to trigger L1 expression due to redundant silencing pathways. Our results illuminate the epigenetic and transcriptional interplay between retrotransposons and their host genome.

p16High senescence restricts cellular plasticity during somatic cell reprogramming.

Despite advances in four-factor (4F)-induced reprogramming (4FR) in vitro and in vivo, how 4FR interconnects with senescence remains largely under investigated. Here, using genetic and chemical approaches to manipulate senescent cells, we show that removal of p16High cells resulted in the 4FR of somatic cells into totipotent-like stem cells. These cells expressed markers of both pluripotency and the two-cell embryonic state, readily formed implantation-competent blastoids and, following morula aggregation, contributed to embryonic and extraembryonic lineages. We identified senescence-dependent regulation of nicotinamide N-methyltransferase as a key mechanism controlling the S-adenosyl-L-methionine levels during 4FR that was required for expression of the two-cell genes and acquisition of an extraembryonic potential. Importantly, a partial 4F epigenetic reprogramming in old mice was able to reverse several markers of liver aging only in conjunction with the depletion of p16High cells. Our results show that the presence of p16High senescent cells limits cell plasticity, whereas their depletion can promote a totipotent-like state and histopathological tissue rejuvenation during 4F reprogramming.

Rapamycin-sensitive signals control TCR/CD28-driven Ifng, Il4 and Foxp3 transcription and promoter region methylation.

The mammalian target of rapamycin (mTOR) controls T-cell differentiation in response to polarizing cytokines. We previously found that mTOR blockade by rapamycin (RAPA) delays the G1-S cell cycle transition and lymphocyte proliferation. Here, we report that both mTOR complex 1 and mTOR complex 2 are readily activated following TCR/CD28 engagement and are critical for early expression of Ifng, Il4 and Foxp3, and for effector T cell differentiation in the absence of polarizing cytokines. While inhibition of mTOR complex 1 and cell division were evident at low doses of RAPA, inhibition of mTOR complex 2, Ifng, Il4 and Foxp3 expression, and T-cell polarization required higher doses and more prolonged treatments. We found that while T-bet and GATA3 were readily induced following TCR/CD28 engagement, administration of RAPA delayed their expression, and interfered with the loss of DNA methylation within Ifng and Il4 promoter regions. In contrast, RAPA prevented activation-dependent DNA methylation of the Foxp3 promoter favoring Foxp3 expression. As a result, RAPA-cultured cells lacked immediate effector functions and instead were enriched for IL-2+ cells. We propose that mTOR-signaling, by timing the expression of critical transcription factors and DNA methylation of proximal promoter regions, regulates transcriptional competence at immunologically relevant sites and hence lymphocyte differentiation.

Two modes of transcriptional activation at native promoters by NF-kappaB p65.

The NF-kappaB family of transcription factors is crucial for the expression of multiple genes involved in cell survival, proliferation, differentiation, and inflammation. The molecular basis by which NF-kappaB activates endogenous promoters is largely unknown, but it seems likely that it should include the means to tailor transcriptional output to match the wide functional range of its target genes. To dissect NF-kappaB-driven transcription at native promoters, we disrupted the interaction between NF-kappaB p65 and the Mediator complex. We found that expression of many endogenous NF-kappaB target genes depends on direct contact between p65 and Mediator, and that this occurs through the Trap-80 subunit and the TA1 and TA2 regions of p65. Unexpectedly, however, a subset of p65-dependent genes are transcribed normally even when the interaction of p65 with Mediator is abolished. Moreover, a mutant form of p65 lacking all transcription activation domains previously identified in vitro can still activate such promoters in vivo. We found that without p65, native NF-kappaB target promoters cannot be bound by secondary transcription factors. Artificial recruitment of a secondary transcription factor was able to restore transcription of an otherwise NF-kappaB-dependent target gene in the absence of p65, showing that the control of promoter occupancy constitutes a second, independent mode of transcriptional activation by p65. This mode enables a subset of promoters to utilize a wide choice of transcription factors, with the potential to regulate their expression accordingly, whilst remaining dependent for their activation on NF-kappaB.

High-Resolution ChIP-MNase Mapping of Nucleosome Positions at Selected Genomic Loci and Alleles.

The positioning of nucleosomes regulates the accessibility of genomic DNA and can impact the activities of functional elements. Nucleosome positioning is highly consistent at each genomic location in any particular cell-type, but can vary in an orchestrated fashion between different cell-types and between genomic loci according to their activities. Here, we describe a technique-"ChIP-MNase" (chromatin immunoprecipitation linked to micrococcal nuclease mapping)-to determine nucleosome positions at chosen sets of genomic features that can be defined by their molecular composition and recovered by chromatin immunoprecipitation. ChIP-MNase enables high-resolution analysis of nucleosome positioning at genomic regions-of-interest and can allow differential analysis of alleles undergoing distinct molecular processes.

Degradation of promoter-bound p65/RelA is essential for the prompt termination of the nuclear factor kappaB response.

Transcription factors of the nuclear factor (NF)-kappaB/Rel family translocate into the nucleus upon degradation of the IkappaBs. Postinduction repression of NF-kappaB activity depends on NF-kappaB-regulated resynthesis of IkappaBalpha, which dissociates NF-kappaB from DNA and exports it to the cytosol. We found that after activation, p65/RelA is degraded by the proteasome in the nucleus and in a DNA binding-dependent manner. If proteasome activity is blocked, NF-kappaB is not promptly removed from some target genes in spite of IkappaBalpha resynthesis and sustained transcription occurs. These results indicate that proteasomal degradation of p65/RelA does not merely regulate its stability and abundance, but also actively promotes transcriptional termination.

Regulation of the chemokine receptor CXCR4 by hypoxia.

Cell adaptation to hypoxia (Hyp) requires activation of transcriptional programs that coordinate expression of genes involved in oxygen delivery (via angiogenesis) and metabolic adaptation (via glycolysis). Here, we describe that oxygen availability is a determinant parameter in the setting of chemotactic responsiveness to stromal-derived factor 1 (CXCL12). Low oxygen concentration induces high expression of the CXCL12 receptor, CXC receptor 4 (CXCR4), in different cell types (monocytes, monocyte-derived macrophages, tumor-associated macrophages, endothelial cells, and cancer cells), which is paralleled by increased chemotactic responsiveness to its specific ligand. CXCR4 induction by Hyp is dependent on both activation of the Hyp-inducible factor 1 alpha and transcript stabilization. In a relay multistep navigation process, the Hyp-Hyp-inducible factor 1 alpha-CXCR4 pathway may regulate trafficking in and out of hypoxic tissue microenvironments.

Role of cell-type specific nucleosome positioning in inducible activation of mammalian promoters.

The organization of nucleosomes across functional genomic elements represents a critical layer of control. Here, we present a strategy for high-resolution nucleosome profiling at selected genomic features, and use this to analyse dynamic nucleosome positioning at inducible and cell-type-specific mammalian promoters. We find that nucleosome patterning at inducible promoters frequently resembles that at active promoters, even before stimulus-driven activation. Accordingly, the nucleosome profile at many inactive inducible promoters is sufficient to predict cell-type-specific responsiveness. Induction of gene expression is generally not associated with major changes to nucleosome patterning, and a subset of inducible promoters can be activated without stable nucleosome depletion from their transcription start sites. These promoters are generally dependent on remodelling enzymes for their inducible activation, and exhibit transient nucleosome depletion only at alleles undergoing transcription initiation. Together, these data reveal how the responsiveness of inducible promoters to activating stimuli is linked to cell-type-specific nucleosome patterning.

P2RX7B is a new theranostic marker for lung adenocarcinoma patients.

Rationale: The characterization of new theranostic biomarkers is crucial to improving the clinical outcome of patients with advanced lung cancer. Here, we aimed at characterizing the P2RX7 receptor, a positive modulator of the anti-tumor immune response, in patients with lung adenocarcinoma. Methods: The expression of P2RX7 and its splice variants was analyzed by RT-qPCR using areas of tumor and non-tumor lung adenocarcinoma (LUAD) tissues on both immune and non-immune cells. The biological activity of P2RX7 was studied by flow cytometry using fluorescent dyes. Bi-molecular fluorescence complementation and confocal microscopy were used to assess the oligomerization of P2RX7. Tumor immune infiltrates were characterized by immunohistochemistry. Results: Fifty-three patients with LUAD were evaluated. P2RX7A, and 3 alternative splice variants were expressed in LUAD tissues and expression was down regulated in tumor versus adjacent non-tumor tissues. The protein retained biological activity only in immune cells. The P2RX7B splice variant was differentially upregulated in immune cells (P < 0.001) of the tumor and strong evidence of oligomerization of P2RX7A and B was observed in the HEK expression model, which correlated with a default in the activity of P2RX7. Finally, LUAD patients with a high level of P2RX7B had non-inflamed tumors (P = 0.001). Conclusion: Our findings identified P2RX7B as a new theranostic tool to restore functional P2RX7 activity and open alternative therapeutic opportunities to improve LUAD patient outcome.

Z and Mmalton-1-antitrypsin deficiency-associated hepatocellular carcinoma: a genetic study.

BACKGROUND: The histological hallmark of alpha-1-antitrypsin deficiency (AATD) is the presence of periodic acid-Schiff diastase (PASD)-resistant positive globules in hepatocytes, with a heterogeneous distribution. It is noteworthy that hepatocellular carcinoma (HCC) arises specifically from the AAT-negative areas but the reason for this remains unclear. AIM: To determine whether the different distribution of AAT globules within neoplastic and non-neoplastic hepatocytes is the result of a self-induced correction of the genetic defect. PATIENTS AND METHODS: Two HCV-positive patients with AATD-associated HCC were studied. One patient harboured a compound heterozygous PiSZ genotype whereas the other showed the rarer PiMMmalton in heterozygosity. In both cases, neoplastic hepatocytes appeared globule devoid, while non-neoplastic hepatocytes showed intracytoplasmic accumulation of PASD-positive globules. Laser-assisted microdissection was used to assess a genotype/phenotype correlation in single liver cells from HCC and from non-neoplastic hepatocytes. RESULTS: Direct sequencing of DNA purified from globule-devoid and globule-filled hepatocytes demonstrated that all liver cells carried the same mutant genetic background. CONCLUSION: Our findings indicate that (i) both variants of HCC arising in AAT deficiency (Z and Mmalton) do not accumulate the mutant protein and (ii) the different phenotypic appearance of hepatocytes is not the result of a retromutation during neoplastic transformation, but other mechanisms should be investigated.

Increased interleukin-10 mRNA stability in melanoma cells is associated with decreased levels of A + U-rich element binding factor AUF1.

Abnormal production of interleukin-10 (IL-10) is observed in some pathologic conditions. For example, compared with normal melanocytes, IL-10 expression is elevated in melanoma cells. IL-10 overexpression could inhibit both immune surveillance and tumor rejection. We investigated a potential posttranscriptional mechanism for IL-10 overexpression in melanoma cells. In normal melanocytes, the half-life of IL-10 mRNA is 7 min, whereas in the melanoma cell line MNT1, the half-life is 75 min. This 10-fold difference could account, at least in part, for IL-10 overexpression in MNT1 cells. Examination of the 3'-untranslated region (3'-UTR) of IL-10 mRNA revealed a suspected A + U-rich element (ARE) that might target the mRNA for rapid degradation. Transfection experiments confirmed that these sequences promote rapid degradation when inserted into a normally stable mRNA, indicating ARE functionality. As AREs act via their interactions with ARE-binding proteins, we examined cytoplasmic proteins from normal melanocytes and MNT1 cells for IL-10 ARE-binding activity. Compared with cytoplasmic extracts of normal melanocytes, cytoplasmic extracts of MNT1 cells possess substantially less ARE-binding activity, consistent with the extended half-life of IL-10 mRNA in MNT1 cells. Finally, we find that the ARE-binding protein AUF1 comprises the major ARE-binding activity in cytoplasmic extracts of normal melanocytes. By contrast, AUF1 is not detectable in cytoplasmic extracts of MNT1 cells but appears restricted to the nuclear fraction. Together, these data suggest a mechanism whereby reduced cytoplasmic levels of AUF1 in MNT1 melanoma cells may lead to IL-10 overexpression, with deleterious consequences for tumor surveillance and rejection.

Cytokine responses in young and old rhesus monkeys: effect of caloric restriction.

Caloric restriction (CR) is the only known intervention demonstrated to retard a great variety of aging processes, extend median and maximum life-span, and decrease the incidence of age-associated diseases in mammals. Paralleling findings from rodent studies, studies in rhesus monkeys (Macaca mulatta) suggest that CR may retard many age-sensitive parameters in primates. A recent study in rhesus monkeys showed age-related dysregulation of cytokine levels. Specifically, age-related increases in interleukin-10 (IL-10) and IL-6 proteins were observed in supernatants from lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs), and interferon-gamma (IFN-gamma) protein exhibited an age-related decrease in phytohemagglutinin (PHA)-stimulated PBMCs. To investigate effects of CR on age-related changes in cytokine production, we obtained PBMCs from control and CR rhesus monkeys aged 6-7 and 22-25 years. We evaluated IL-10 and IL-6 protein and gene expression after exposure to LPS and IFN-gamma protein and gene expression after PHA stimulation. The results revealed significantly higher levels of IFN-gamma protein and gene expression in aged monkeys on CR for 2 years compared with controls. No significant CR effects were observed on IL-10 and IL-6 protein levels. IFN-gamma plays an important role in the initial defense mechanism against viral and microbial disease and cancer. Altered regulation of IFN-gamma in old CR rhesus monkeys may be a key factor in reducing cancer incidence and other age-associated diseases.

TNF signaling: key protocols.

Tumor necrosis factor (TNF) is a pleiotropic cytokine that signals inflammation as well as cell death. We focus herein on the inflammatory pathway, giving particular emphasis to the in vitro methods used to study intracellular signaling mediators. The signal transduction cascade that TNF triggers after binding to the TNF receptors (TNFRs), flows throughout a series of protein-protein interactions as well as kinase activations, and finally leads to the translocation of distinct transcription factors within the nucleus, eventually inducing the transcription of specific inflammatory genes. In this chapter, we describe the analysis of the TNF receptor signaling complex by immunoprecipitation (IP), the activation of different MAP kinases by Western blot, the analysis of transcription factor activation by either electrophoretic mobility-shift assay (EMSA) or reporter assay, and the analysis of TNF-induced genes by chromatin IP (ChIP).

Heterochromatin boundaries are hotspots for de novo kinetochore formation.

The centromere-specific histone H3 variant CENH3 (also known as CENP-A) is considered to be an epigenetic mark for establishment and propagation of centromere identity. Pulse induction of CENH3 (Drosophila CID) in Schneider S2 cells leads to its incorporation into non-centromeric regions and generates CID islands that resist clearing from chromosome arms for multiple cell generations. We demonstrate that CID islands represent functional ectopic kinetochores, which are non-randomly distributed on the chromosome and show a preferential localization near telomeres and pericentric heterochromatin in transcriptionally silent, intergenic chromatin domains. Although overexpression of heterochromatin protein 1 (HP1) or increasing histone acetylation interferes with CID island formation on a global scale, induction of a locally defined region of synthetic heterochromatin by targeting HP1-LacI fusions to stably integrated Lac operator arrays produces a proximal hotspot for CID deposition. These data indicate that the characteristics of regions bordering heterochromatin promote de novo kinetochore assembly and thereby contribute to centromere identity.