Patrick A. Navas

The accessible chromatin landscape of the human genome.

DNase I hypersensitive sites (DHSs) are markers of regulatory DNA and have underpinned the discovery of all classes of cis-regulatory elements including enhancers, promoters, insulators, silencers and locus control regions. Here we present the first extensive map of human DHSs identified through genome-wide profiling in 125 diverse cell and tissue types. We identify ∼2.9 million DHSs that encompass virtually all known experimentally validated cis-regulatory sequences and expose a vast trove of novel elements, most with highly cell-selective regulation. Annotating these elements using ENCODE data reveals novel relationships between chromatin accessibility, transcription, DNA methylation and regulatory factor occupancy patterns. We connect ∼580,000 distal DHSs with their target promoters, revealing systematic pairing of different classes of distal DHSs and specific promoter types. Patterning of chromatin accessibility at many regulatory regions is organized with dozens to hundreds of co-activated elements, and the transcellular DNase I sensitivity pattern at a given region can predict cell-type-specific functional behaviours. The DHS landscape shows signatures of recent functional evolutionary constraint. However, the DHS compartment in pluripotent and immortalized cells exhibits higher mutation rates than that in highly differentiated cells, exposing an unexpected link between chromatin accessibility, proliferative potential and patterns of human variation.

Developmental specificity of the interaction between the locus control region and embryonic or fetal globin genes in transgenic mice with an HS3 core deletion.

ABSTRACT The human β-globin locus control region (LCR) consists of five erythroid-lineage-specific DNase I-hypersensitive sites (HSs) and is required for activation of the β-globin locus chromatin domain and globin gene expression. Each DNase I-HS of the LCR consists of a highly conserved core element and flanking sequences. To analyze the functional role of the core elements of the HSs, we deleted a 234-bp fragment encompassing the core of HS3 (HS3c) from a β-globin locus residing on a 248-kb β-locus yeast artificial chromosome and analyzed its function in F2 progeny of transgenic mice. Human ɛ-globin gene expression was absent at day 10 and severely reduced in the day 12 embryonic erythropoiesis of mice lacking HS3c. In contrast, γ-globin gene expression was normal in embryonic erythropoiesis but it was absent in definitive erythropoiesis in the fetal liver. These results indicate that the core element of HS3 is necessary for ɛ-globin gene transcription in embryonic cells and for γ-globin gene transcription in definitive cells. Normal γ-globin gene expression in embryonic cells and the absence of γ-globin gene expression in definitive cells show that different HSs interact with γ-globin gene promoters in these two stages of development. Such results provide direct evidence for developmental stage specificity of the interactions between the core elements of HSs and the promoters of the globin genes.

Genomic discovery of potent chromatin insulators for human gene therapy

Insertional mutagenesis and genotoxicity, which usually manifest as hematopoietic malignancy, represent major barriers to realizing the promise of gene therapy. Although insulator sequences that block transcriptional enhancers could mitigate or eliminate these risks, so far no human insulators with high functional potency have been identified. Here we describe a genomic approach for the identification of compact sequence elements that function as insulators. These elements are highly occupied by the insulator protein CTCF, are DNase I hypersensitive and represent only a small minority of the CTCF recognition sequences in the human genome. We show that the elements identified acted as potent enhancer blockers and substantially decreased the risk of tumor formation in a cancer-prone animal model. The elements are small, can be efficiently accommodated by viral vectors and have no detrimental effects on viral titers. The insulators we describe here are expected to increase the safety of gene therapy for genetic diseases.

Synergistic and Additive Properties of the Beta-Globin Locus Control Region (LCR) Revealed by 5′HS3 Deletion Mutations: Implication for LCR Chromatin Architecture

ABSTRACT Deletion of the 234-bp core element of the DNase I hypersensitive site 3 (5′HS3) of the locus control region (LCR) in the context of a human beta-globin locus yeast artificial chromosome (β-YAC) results in profound effects on globin gene expression in transgenic mice. In contrast, deletion of a 2.3-kb 5′HS3 region, which includes the 234-bp core sequence, has a much milder phenotype. Here we report the effects of these deletions on chromatin structure in the beta-globin locus of adult erythroblasts. The 234-bp 5′HS3 deletion abolished histone acetylation throughout the β-globin locus; recruitment of RNA polymerase II (pol II) to the LCR and beta-globin gene promoter was reduced to a basal level; and formation of all the 5′ DNase I hypersensitive sites of the LCR was disrupted. The 2.3-kb 5′HS3 deletion mildly reduced the level of histone acetylation but did not change the profile across the whole locus; the 5′ DNase I hypersensitive sites of the LCR were formed, but to a lesser extent; and recruitment of pol II was reduced, but only marginally. These data support the hypothesis that the LCR forms a specific chromatin structure and acts as a single entity. Based on these results we elaborate on a model of LCR chromatin architecture which accommodates the distinct phenotypes of the 5′HS3 and HS3 core deletions.

γ-Globin Gene Expression in Chemical Inducer of Dimerization (CID)-dependent Multipotential Cells Established from Human β-Globin Locus Yeast Artificial Chromosome (β-YAC) Transgenic Mice

Identification of trans-acting factors or drugs capable of reactivating γ-globin gene expression is complicated by the lack of suitable cell lines. Human K562 cells co-express ϵ- and γ-globin but not β-globin; transgenic mouse erythroleukemia 585 cells express predominantly human β-globin but also γ-globin; and transgenic murine GM979 cells co-express human γ-and β-globin. Human β-globin locus yeast artificial chromosome transgenic mice display correct developmental regulation of β-like globin gene expression. We rationalized that cells established from the adult bone marrow of these mice might express exclusively β-globin and therefore could be employed to select or screen inducers of γ-globin expression. A thrombopoietin receptor derivative that brings the proliferative status of primary mouse bone marrow cells under control of a chemical inducer of dimerization was employed to institute and maintain these cell populations. Human β-globin was expressed, but γ-globin was not; a similar expression pattern was observed in cells derived from fetal liver. γ-Globin expression was induced upon exposure to 5-azacytidine, in cells derived from –117 Greek hereditary persistence of fetal hemoglobin human β-globin locus yeast artificial chromosome (β-YAC) mice, showing that the hereditary persistence of fetal hemoglobin (HPFH) phenotype was maintained in these cells or was reactivated by an artificial zinc finger-γ-globin transcription factor and the previously identified fetal globin transactivators fetal Krüppel-like factor (FKLF) and fetal globin-increasing factor (FGIF). These cells may be useful for identifying transcription factors that reactivate γ-globin synthesis or screening γ-globin inducers for the treatment of sickle cell disease or β-thalassemia.

Genome architecture of the human β-globin locus affects developmental regulation of gene expression

ABSTRACT To test the role of gene order in globin gene expression, mutant human β-globin locus yeast artificial chromosome constructs were used, each having one additional globin gene encoding a “marked” transcript (εm, γm, or βm) integrated at different locations within the locus. When a βm-globin gene was placed between the locus control region (LCR) and the ε-globin gene, βm-globin expression dominated primitive and definitive erythropoiesis; only βm-globin mRNA was detected during the fetal and adult definitive stages of erythropoiesis. When an Aγm-globin gene was placed at the same location, Aγm-globin was expressed during embryonic erythropoiesis and the fetal liver stage of definitive erythropoiesis but was silenced during the adult stage. The downstream wild-type γ-globin genes were not expressed. When an εm-globin gene was placed between the δ- and β-globin genes, it remained silent during embryonic erythropoiesis; only the LCR-proximal wild-type ε-globin gene was expressed. Placement of a βm-globin gene upstream of the Gγ-globin gene resulted in expression of βm-globin in embryonic cells and in a significant decrease in expression of the downstream wild-type β-globin gene. These results indicate that distance from the LCR, an inherent property of spatial gene order, is a major determinant of temporal gene expression during development.

A European population in Minoan Bronze Age Crete

The first advanced Bronze Age civilization of Europe was established by the Minoans about 5,000 years before present. Since Sir Arthur Evans exposed the Minoan civic centre of Knossos, archaeologists have speculated on the origin of the founders of the civilization. Evans proposed a North African origin; Cycladic, Balkan, Anatolian and Middle Eastern origins have also been proposed. Here we address the question of the origin of the Minoans by analysing mitochondrial DNA from Minoan osseous remains from a cave ossuary in the Lassithi plateau of Crete dated 4,400–3,700 years before present. Shared haplotypes, principal component and pairwise distance analyses refute the Evans North African hypothesis. Minoans show the strongest relationships with Neolithic and modern European populations and with the modern inhabitants of the Lassithi plateau. Our data are compatible with the hypothesis of an autochthonous development of the Minoan civilization by the descendants of the Neolithic settlers of the island.

Genetic origins of the Minoans and Mycenaeans

The origins of the Bronze Age Minoan and Mycenaean cultures have puzzled archaeologists for more than a century. We have assembled genome-wide data from 19 ancient individuals, including Minoans from Crete, Mycenaeans from mainland Greece, and their eastern neighbours from southwestern Anatolia. Here we show that Minoans and Mycenaeans were genetically similar, having at least three-quarters of their ancestry from the first Neolithic farmers of western Anatolia and the Aegean, and most of the remainder from ancient populations related to those of the Caucasus and Iran. However, the Mycenaeans differed from Minoans in deriving additional ancestry from an ultimate source related to the hunter–gatherers of eastern Europe and Siberia, introduced via a proximal source related to the inhabitants of either the Eurasian steppe or Armenia. Modern Greeks resemble the Mycenaeans, but with some additional dilution of the Early Neolithic ancestry. Our results support the idea of continuity but not isolation in the history of populations of the Aegean, before and after the time of its earliest civilizations.

β‐YAC Transgenic Mice for Studying LCR Functiona

Abstract: We have developed methods to produce transgenic mice using yeast artificial chromosomes (YACs) and have applied these methods to the analysis of globin gene regulation using 248 kb β‐globin locus YACs (β‐YACs). The advantages of YAC transgenics are: 1) developmental regulation can be studied in the context of the whole locus, 2) mutations may be readily introduced into the YAC, and 3) the effect of these mutations on gene expression can be analyzed. Mice containing the wild‐type β‐YAC show proper regulation of globin gene expression during development. Transgenics carrying a β‐YAC bearing a −117 Aγ mutation showed the anticipated phenotype of Greek HPFH, demonstrating that mutant β‐YACs can be used to generate mice that recreate human globin developmental mutants. Transgenic mice with YACs have also been used to examine the function of the LCR. Transgenic mice were generated with a β‐YAC containing a deletion of LCR DNAse I‐hypersensitive site 3 (5′HS3). Our results suggest that: 1) the LCR contains functionally redundant elements, 2) the formation of a LCR complex does not require all of the HSs, 3) the individual HSs may modulate the interaction of the LCR with specific globin genes during development, and 4) that most of the HS activity is confined to the core region.

The position of integration affects expression of the Aγ-globin-encoding gene linked to HS3 in transgenic mice

Proper expression of the human beta-globin (beta Glb) locus is dependent on the presence of a major regulatory element located upstream from the beta Glb gene cluster, the locus control region (LCR). The LCR, as well as the individual DNase-I-hypersensitive sites from which it is composed, have been shown to provide position-of-integration-independent expression in transgenic mice. Here, we report that a transgenic founder carrying multiple integrations of a hypersensitive site 3::A gamma globin gene (HS3::A gamma) construct produced three types of progeny, one with zero A gamma expression in the adult stage, one with minimal A gamma expression (1% of A gamma-expressing cells) and one with abundant A gamma expression (100% A gamma-expressing cells). The possibility that these phenotypes were due to parental imprinting or to DNA rearrangements of the transgene or to point mutations of the HS3 core or the A gamma promoter were excluded. The pattern of inheritance of the three HS3::A gamma transgene phenotypes indicate that the transgene has integrated into three different chromosomes. These results provide direct evidence that the HS3 of the LCR is not sufficient to protect the A gamma gene from position effects excerted by the surrounding chromatin.