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.

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.

Long-range control of T-cell development.

In this issue of Blood, Li et al reveal the genetic elements that control the activity of Bcl11b, a critical regulator of T-cell development. Lineage-defining transcription factors (TFs), such as Bcl11b, control key steps in cellular differentiation throughout development, and understanding how these TFs are themselves regulated represents a major challenge.

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.

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.

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.

A metabolic interplay coordinated by HLX regulates myeloid differentiation and AML through partly overlapping pathways.

The H2.0-like homeobox transcription factor (HLX) regulates hematopoietic differentiation and is overexpressed in Acute Myeloid Leukemia (AML), but the mechanisms underlying these functions remain unclear. We demonstrate here that HLX overexpression leads to a myeloid differentiation block both in zebrafish and human hematopoietic stem and progenitor cells (HSPCs). We show that HLX overexpression leads to downregulation of genes encoding electron transport chain (ETC) components and upregulation of PPARδ gene expression in zebrafish and human HSPCs. HLX overexpression also results in AMPK activation. Pharmacological modulation of PPARδ signaling relieves the HLX-induced myeloid differentiation block and rescues HSPC loss upon HLX knockdown but it has no effect on AML cell lines. In contrast, AMPK inhibition results in reduced viability of AML cell lines, but minimally affects myeloid progenitors. This newly described role of HLX in regulating the metabolic state of hematopoietic cells may have important therapeutic implications.

Activation of individual L1 retrotransposon instances is restricted to cell-type dependent permissive loci.

LINE-1 (L1) retrotransposons represent approximately one sixth of the human genome, but only the human-specific L1HS-Ta subfamily acts as an endogenous mutagen in modern humans, reshaping both somatic and germline genomes. Due to their high levels of sequence identity and the existence of many polymorphic insertions absent from the reference genome, the transcriptional activation of individual genomic L1HS-Ta copies remains poorly understood. Here we comprehensively mapped fixed and polymorphic L1HS-Ta copies in 12 commonly-used somatic cell lines, and identified transcriptional and epigenetic signatures allowing the unambiguous identification of active L1HS-Ta copies in their genomic context. Strikingly, only a very restricted subset of L1HS-Ta loci - some being polymorphic among individuals - significantly contributes to the bulk of L1 expression, and these loci are differentially regulated among distinct cell lines. Thus, our data support a local model of L1 transcriptional activation in somatic cells, governed by individual-, locus-, and cell-type-specific determinants.

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.

The mediator complex functions as a coactivator for GATA-1 in erythropoiesis via subunit Med1/TRAP220.

The Mediator complex forms the bridge between transcriptional activators and RNA polymerase II. Mediator subunit Med1/TRAP220 is a key component of Mediator originally found to associate with nuclear hormone receptors. Med1 deficiency causes lethality at embryonic day 11.5 because of defects in heart and placenta development. Here we show that Med1-deficient 10.5 days postcoitum embryos are anemic but have normal numbers of hematopoietic progenitor cells. Med1-deficient progenitor cells have a defect in forming erythroid burst-forming units (BFU-E) and colony-forming units (CFU-E), but not in forming myeloid colonies. At the molecular level, we demonstrate that Med1 interacts physically with the erythroid master regulator GATA-1. In transcription assays, Med1 deficiency leads to a defect in GATA-1-mediated transactivation. In chromatin immunoprecipitation experiments, we find Mediator components at GATA-1-occupied enhancer sites. Thus, we conclude that Mediator subunit Med1 acts as a pivotal coactivator for GATA-1 in erythroid development.