Genomic context sensitizes regulatory elements to genetic disruption.

Genomic context critically modulates regulatory function but is difficult to manipulate systematically. The murine insulin-like growth factor 2 (Igf2)/H19 locus is a paradigmatic model of enhancer selectivity, whereby CTCF occupancy at an imprinting control region directs downstream enhancers to activate either H19 or Igf2. We used synthetic regulatory genomics to repeatedly replace the native locus with 157-kb payloads, and we systematically dissected its architecture. Enhancer deletion and ectopic delivery revealed previously uncharacterized long-range regulatory dependencies at the native locus. Exchanging the H19 enhancer cluster with the Sox2 locus control region (LCR) showed that the H19 enhancers relied on their native surroundings while the Sox2 LCR functioned autonomously. Analysis of regulatory DNA actuation across cell types revealed that these enhancer clusters typify broader classes of context sensitivity genome wide. These results show that unexpected dependencies influence even well-studied loci, and our approach permits large-scale manipulation of complete loci to investigate the relationship between regulatory architecture and function.

Lentiviral vector ALS20 yields high hemoglobin levels with low genomic integrations for treatment of beta-globinopathies.

Ongoing clinical trials for treatment of beta-globinopathies by gene therapy involve the transfer of the beta-globin gene, which requires integration of three to four copies per genome in most target cells. This high proviral load may increase genome toxicity, potentially limiting the safety of this therapy and relegating its use to total body myeloablation. We hypothesized that introducing an additional hypersensitive site from the locus control region, the complete sequence of the second intron of the beta-globin gene, and the ankyrin insulator may enhance beta-globin expression. We identified a construct, ALS20, that synthesized significantly higher adult hemoglobin levels than those of other constructs currently used in clinical trials. These findings were confirmed in erythroblastic cell lines and in primary cells isolated from sickle cell disease patients. Bone marrow transplantation studies in beta-thalassemia mice revealed that ALS20 was curative at less than one copy per genome. Injection of human CD34+ cells transduced with ALS20 led to safe, long-term, and high polyclonal engraftment in xenograft experiments. Successful treatment of beta-globinopathies with ALS20 could potentially be achieved at less than two copies per genome, minimizing the risk of cytotoxic events and lowering the intensity of myeloablation.

A Sir2-regulated locus control region in the recombination enhancer of Saccharomyces cerevisiae specifies chromosome III structure.

The NAD+-dependent histone deacetylase Sir2 was originally identified in Saccharomyces cerevisiae as a silencing factor for HML and HMR, the heterochromatic cassettes utilized as donor templates during mating-type switching. MATa cells preferentially switch to MATα using HML as the donor, which is driven by an adjacent cis-acting element called the recombination enhancer (RE). In this study we demonstrate that Sir2 and the condensin complex are recruited to the RE exclusively in MATa cells, specifically to the promoter of a small gene within the right half of the RE known as RDT1. We also provide evidence that the RDT1 promoter functions as a locus control region (LCR) that regulates both transcription and long-range chromatin interactions. Sir2 represses RDT1 transcription until it is removed from the promoter in response to a dsDNA break at the MAT locus induced by HO endonuclease during mating-type switching. Condensin is also recruited to the RDT1 promoter and is displaced upon HO induction, but does not significantly repress RDT1 transcription. Instead condensin appears to promote mating-type donor preference by maintaining proper chromosome III architecture, which is defined by the interaction of HML with the right arm of chromosome III, including MATa and HMR. Remarkably, eliminating Sir2 and condensin recruitment to the RDT1 promoter disrupts this structure and reveals an aberrant interaction between MATa and HMR, consistent with the partially defective donor preference for this mutant. Global condensin subunit depletion also impairs mating-type switching efficiency and donor preference, suggesting that modulation of chromosome architecture plays a significant role in controlling mating-type switching, thus providing a novel model for dissecting condensin function in vivo.

Targeted deletion of 5'HS2 enhancer of ß-globin locus control region in K562 cells.

Human ß-thalassemia is closely associated with aberrant expression of ß-like globin genes. Human ß-like globin genes are organized in the order of 5'-ε-Gγ-Aγ-δ-ß-3' within the ß-globin locus. The expression of ß-like globin genes is regulated by 3'HS1 and five DNase I hypersensitive sites (5'HS5~5'HS1) in a locus control region. The 5'HS2 enhancer transcribes enhancer RNA and regulates the expression of ε-globin, γ-globin and ß-globin. To further study the function of 5'HS2, we detected the local 3D genomic architecture via chromatin conformation capture experiments and used CRISPR/ Cas9-based DNA fragment editing to delete 5'HS2 in human K562 leukaemia cells. In this study, we found that 5'HS2-mediated chromatin interactions were enriched in a topologically associated domain that was bordered by 3'HS1 and 5'HS5. Within this topologically associated domain, 5'HS2 is highly close to the promoter regions of HBE1, HBG2 and HBG1. Upon deletion of the 5'HS2 enhancer, 91 genes were significantly down-regulated with reduced abundance of H3K27ac at their promoter regions. These down-regulated genes are mainly associated with oxygen transport, immune response, cell adhesion, anti-oxidant and thrombosis. These data suggested that many genes associated with functions of erythrocytes were decreased at transcriptional levels upon deletion of the 5'HS2 enhancer.

Improved Titer and Gene Transfer by Lentiviral Vectors Using Novel, Small ß-Globin Locus Control Region Elements.

ß-globin lentiviral vectors (ß-LV) have faced challenges in clinical translation for gene therapy of sickle cell disease (SCD) due to low titer and sub-optimal gene transfer to hematopoietic stem and progenitor cells (HSPCs). To overcome the challenge of preserving efficacious expression while increasing vector performance, we used published genomic and epigenomic data available through ENCODE to redefine enhancer element boundaries of the ß-globin locus control region (LCR) to construct novel ENCODE core sequences. These novel LCR elements were used to design a ß-LV of reduced proviral length, termed CoreGA-AS3-FB, produced at higher titers and possessing superior gene transfer to HSPCs when compared to the full-length parental ß-LV at equal MOI. At low vector copy number, vectors containing the ENCODE core sequences were capable of reversing the sickle phenotype in a mouse model of SCD. These studies provide a ß-LV that will be beneficial for gene therapy of SCD by significantly reducing the cost of vector production and extending the vector supply.

LDB1-mediated enhancer looping can be established independent of mediator and cohesin.

Mechanistic studies in erythroid cells indicate that LDB1, as part of a GATA1/TAL1/LMO2 complex, brings erythroid-expressed genes into proximity with enhancers for transcription activation. The role of co-activators in establishing this long-range interaction is poorly understood. Here we tested the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and cohesin to establishment of locus control region (LCR)/ß-globin proximity. CRISPR/Cas9 editing of the ß-globin promoter to eliminate the RNA Pol II PIC by deleting the TATA-box resulted in loss of transcription, but enhancer-promoter interaction was unaffected. Additional deletion of the promoter GATA1 site eliminated LDB1 complex and mediator occupancy and resulted in loss of LCR/ß-globin proximity. To separate the roles of LDB1 and mediator in LCR looping, we expressed a looping-competent but transcription-activation deficient form of LDB1 in LDB1 knock down cells: LCR/ß-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the ß-globin promoter forced LCR loop formation in the absence of mediator or cohesin occupancy. Moreover, ENCODE data and our chromatin immunoprecipitation results indicate that cohesin is almost completely absent from validated and predicted LDB1-regulated erythroid enhancer-gene pairs. Thus, lineage specific factors largely mediate enhancer-promoter looping in erythroid cells independent of mediator and cohesin.

[Structural and Functional Organization of the Mitochondrial DNA Control Region in the Woolly Mammoth (Mammuthus primigenius)].

The woolly mammoth mitochondrial genome (including the Malolyakhovsky mammoth) has been previously sequenced, followed by the annotation of all its genes (MF770243). In this study, based on the Malolyakhovsky mammoth, we describe for the first time the sites of functional significance in the control region of the woolly mammoth mitogenome.

CTCF-binding elements 1 and 2 in the Igh intergenic control region cooperatively regulate V(D)J recombination.

Ig heavy chain (IgH) variable region exons are assembled from V, D, and J gene segments during early B-lymphocyte differentiation. A several megabase region at the "distal" end of the mouse IgH locus (Igh) contains hundreds of V(H)s, separated by an intergenic region from Igh Ds, J(H)s, and constant region exons. Diverse primary Igh repertoires are generated by joining Vs, Ds, and Js in different combinations, with a given B cell productively assembling only one combination. The intergenic control region 1 (IGCR1) in the V(H)-to-D intergenic region regulates Igh V(D)J recombination in the contexts of developmental order, lineage specificity, and feedback from productive rearrangements. IGCR1 also diversifies IgH repertoires by balancing proximal and distal V(H) use. IGCR1 functions in all these regulatory contexts by suppressing predominant rearrangement of D-proximal V(H)s. Such IGCR1 functions were neutralized by simultaneous mutation of two CCCTC-binding factor (CTCF)-binding elements (CBE1 and CBE2) within it. However, it was unknown whether only one CBE mediates IGCR1 functions or whether both function in this context. To address these questions, we generated mice in which either IGCR1 CBE1 or CBE2 was replaced with scrambled sequences that do not bind CTCF. We found that inactivation of CBE1 or CBE2 individually led to only partial impairment of various IGCR1 functions relative to the far greater effects of inactivating both binding elements simultaneously, demonstrating that they function cooperatively to achieve full IGCR1 regulatory activity. Based on these and other findings, we propose an orientation-specific looping model for synergistic CBE1 and CBE2 functions.

Using ancient DNA to study the origins and dispersal of ancestral Polynesian chickens across the Pacific.

The human colonization of Remote Oceania remains one of the great feats of exploration in history, proceeding east from Asia across the vast expanse of the Pacific Ocean. Human commensal and domesticated species were widely transported as part of this diaspora, possibly as far as South America. We sequenced mitochondrial control region DNA from 122 modern and 22 ancient chicken specimens from Polynesia and Island Southeast Asia and used these together with Bayesian modeling methods to examine the human dispersal of chickens across this area. We show that specific techniques are essential to remove contaminating modern DNA from experiments, which appear to have impacted previous studies of Pacific chickens. In contrast to previous reports, we find that all ancient specimens and a high proportion of the modern chickens possess a group of unique, closely related haplotypes found only in the Pacific. This group of haplotypes appears to represent the authentic founding mitochondrial DNA chicken lineages transported across the Pacific, and allows the early dispersal of chickens across Micronesia and Polynesia to be modeled. Importantly, chickens carrying this genetic signature persist on several Pacific islands at high frequencies, suggesting that the original Polynesian chicken lineages may still survive. No early South American chicken samples have been detected with the diagnostic Polynesian mtDNA haplotypes, arguing against reports that chickens provide evidence of Polynesian contact with pre-European South America. Two modern specimens from the Philippines carry haplotypes similar to the ancient Pacific samples, providing clues about a potential homeland for the Polynesian chicken.

The New -474(C→T) Substitution Discovered in the HBG2 Promoter of a Sardinian δß-Thalassemia Carrier.

During a screening for hemoglobinopathies, we found a carrier of the Sardinian δß-thalassemia condition. The proband's hematology and hemoglobin (Hb) profile agreed with those of the other carriers previously identified during our diagnostic program except for the fetal Hb (HbF) composition, which consisted of both α2Aγ2 and α2Gγ2 instead of nearly 100% α2Aγ2. In order to explain the unusual γ-chain ratio, sequencing of the Gγ promoter was carried out and revealed two nucleotide substitutions in cis: C→T at position -474 and A→G at position -309 from the Cap site. The latter had previously been observed in subjects with raised HbF levels, although it has not yet been evaluated at functional level. We used the luciferase assay to determine whether the two mutations modify the transcriptional activity of the Gγ promoter. Results indicated that the observed in vivo Gγ-globin production cannot be translated into increased in vitro promoter function, suggesting that the assessed mutations cannot be considered as functional single nucleotide polymorphisms per se; instead, a more complex regulatory mechanism might be involved.

Transcription factor YY1 is essential for regulation of the Th2 cytokine locus and for Th2 cell differentiation.

The Th2 locus control region (LCR) has been shown to be important in efficient and coordinated cytokine gene regulation during Th2 cell differentiation. However, the molecular mechanism for this is poorly understood. To study the molecular mechanism of the Th2 LCR, we searched for proteins binding to it. We discovered that transcription factor YY1 bound to the LCR and the entire Th2 cytokine locus in a Th2-specific manner. Retroviral overexpression of YY1 induced Th2 cytokine expression. CD4-specific knockdown of YY1 in mice caused marked reduction in Th2 cytokine expression, repressed chromatin remodeling, decreased intrachromosomal interactions, and resistance in an animal model of asthma. YY1 physically associated with GATA-binding protein-3 (GATA3) and is required for GATA3 binding to the locus. YY1 bound to the regulatory elements in the locus before GATA3 binding. Thus, YY1 cooperates with GATA3 and is required for regulation of the Th2 cytokine locus and Th2 cell differentiation.

Hypersensitive site 6 of the Th2 locus control region is essential for Th2 cytokine expression.

The T helper type 2 (Th2) cytokine genes Il4, Il5, and Il13 are contained within a 140-kb region of mouse chromosome 11 and their expression is controlled by a locus control region (LCR) embedded within this locus. The LCR is composed of a number of DNase I-hypersensitive sites (HSs), which are believed to encompass the regulatory core of the LCR. To determine the function of these sites, mutant mice were generated in which combinations of these HSs had been deleted from the endogenous LCR, and the effect on Th2 cytokine expression was assessed through the use of in vivo and in vitro models. These experiments revealed that, although all of the hypersensitive sites analyzed are important for appropriate LCR function, some sites are more important than others in regulating cytokine expression. Interestingly, each LCR mutation showed contrasting effects on cytokine expression, in some cases with mutants displaying opposing phenotypes between in vitro cultures and in vivo immunizations. These studies indicated that Rad50 hypersensitive site 6 was the singularly most important HS for Th2 cytokine expression, displaying consistent reductions in cytokine levels in all models tested. Furthermore analysis of chromatin modifications revealed that deletion of Rad50 hypersensitive site 6 impacted epigenetic modifications at the promoters of the Il4, Il5, and Il13 genes as well as other regulatory sites within the Th2 locus.

R-loop formation at Snord116 mediates topotecan inhibition of Ube3a-antisense and allele-specific chromatin decondensation.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are oppositely imprinted autism-spectrum disorders with known genetic bases, but complex epigenetic mechanisms underlie their pathogenesis. The PWS/AS locus on 15q11-q13 is regulated by an imprinting control region that is maternally methylated and silenced. The PWS imprinting control region is the promoter for a one megabase paternal transcript encoding the ubiquitous protein-coding Snrpn gene and multiple neuron-specific noncoding RNAs, including the PWS-related Snord116 repetitive locus of small nucleolar RNAs and host genes, and the antisense transcript to AS-causing ubiquitin ligase encoding Ube3a (Ube3a-ATS). Neuron-specific transcriptional progression through Ube3a-ATS correlates with paternal Ube3a silencing and chromatin decondensation. Interestingly, topoisomerase inhibitors, including topotecan, were recently identified in an unbiased drug screen for compounds that could reverse the silent paternal allele of Ube3a in neurons, but the mechanism of topotecan action on the PWS/AS locus is unknown. Here, we demonstrate that topotecan treatment stabilizes the formation of RNA:DNA hybrids (R loops) at G-skewed repeat elements within paternal Snord116, corresponding to increased chromatin decondensation and inhibition of Ube3a-ATS expression. Neural precursor cells from paternal Snord116 deletion mice exhibit increased Ube3a-ATS levels in differentiated neurons and show a reduced effect of topotecan compared with wild-type neurons. These results demonstrate that the AS candidate drug topotecan acts predominantly through stabilizing R loops and chromatin decondensation at the paternally expressed PWS Snord116 locus. Our study holds promise for targeted therapies to the Snord116 locus for both AS and PWS.

Spatially differentiated expression of quadruplicated green-sensitive RH2 opsin genes in zebrafish is determined by proximal regulatory regions and gene order to the locus control region.

BACKGROUND: Fish are remarkably diverse in repertoires of visual opsins by gene duplications. Differentiation of their spatiotemporal expression patterns and absorption spectra enables fine-tuning of feature detection in spectrally distinct regions of the visual field during ontogeny. Zebrafish have quadruplicated green-sensitive (RH2) opsin genes in tandem (RH2-1, -2, -3, -4), which are expressed in the short member of the double cones (SDC). The shortest wavelength RH2 subtype (RH2-1) is expressed in the central to dorsal area of the adult retina. The second shortest wave subtype (RH2-2) is expressed overlapping with RH2-1 but extending outside of it. The second longest wave subtype (RH2-3) is expressed surrounding the RH2-2 area, and the longest wave subtype (RH2-4) is expressed outside of the RH2-3 area broadly occupying the ventral area. Expression of the four RH2 genes in SDC requires a single enhancer (RH2-LCR), but the mechanism of their spatial differentiation remains elusive. RESULTS: Functional comparison of the RH2-LCR with its counterpart in medaka revealed that the regulatory role of the RH2-LCR in SDC-specific expression is evolutionarily conserved. By combining the RH2-LCR and the proximal upstream region of each RH2 gene with fluorescent protein reporters, we show that the RH2-LCR and the RH2-3 proximal regulatory region confer no spatial selectivity of expression in the retina. But those of RH2-1, -2 and -4 are capable of inducing spatial differentiation of expression. Furthermore, by analyzing transgenic fish with a series of arrays consisting of the RH2-LCR and multiple upstream regions of the RH2 genes in different orders, we show that a gene expression pattern related to an upstream region is greatly influenced by another flanking upstream region in a relative position-dependent manner. CONCLUSIONS: The zebrafish RH2 genes except RH2-3 acquired differential cis-elements in the proximal upstream regions to specify the differential expression patterns. The input from these proximal elements collectively dictates the actual gene expression pattern of the locus, context-dependently. Importantly, competition for the RH2-LCR activity among the replicates is critical in this collective regulation, facilitating differentiation of expression among them. This combination of specificity and generality enables seemingly complicated spatial differentiation of duplicated opsin genes characteristic in fish.

Genetic analysis of captive proboscis monkeys.

Information on the genetic relationships of captive founders is important for captive population management. In this study, we investigated DNA polymorphisms of four microsatellite loci and the mitochondrial control region sequence of five proboscis monkeys residing in a Japanese zoo as captive founders, to clarify their genetic relationship. We found that two of the five monkeys appeared to be genetically related. Furthermore, the haplotypes of the mitochondrial control region of the five monkeys were well differentiated from the haplotypes previously reported from wild populations from the northern area of Borneo, indicating a greater amount of genetic diversity in proboscis monkeys than previously reported.

Heterozygosity for deletion of hypersensitive site 3 in the human locus control region has an unexpected minor effect on red cell phenotype.

The locus control region (LCR) is a genetic region that regulates the expression of the ß-globin locus (HBB locus). This region is composed of several DNase I hypersensitive sites (HSs) in which the regulatory functions of the LCR may reside. To date, some individuals bearing deletions of several HSs or even the complete LCR have been described. Although the globin genes of the HBB locus are intact, most of these patients suffer thalassemia due to the reduced expression of such genes. The LCR and the HSs forming it have been thoroughly studied in different genetic models. However, seemingly contradictory results are often obtained. Here, we describe the first deletion found in humans exclusively affecting the HS3 element of the LCR. The adult carrying this deletion shows very mild hematological modifications, indicating that HS3 deletion does not severely impair the ß-gene expression. Our results also reveal limitations of the murine models when studying the native mouse genes for understanding human diseases like thalassemias.

The hypersensitive sites of the murine ß-globin locus control region act independently to affect nuclear localization and transcriptional elongation.

The ß-globin locus control region (LCR) is necessary for high-level ß-globin gene transcription and differentiation-dependent relocation of the ß-globin locus from the nuclear periphery to the central nucleoplasm and to foci of hyperphosphorylated Pol II "transcription factories" (TFys). To determine the contribution of individual LCR DNaseI hypersensitive sites (HSs) to transcription and nuclear location, in the present study, we compared ß-globin gene activity and location in erythroid cells derived from mice with deletions of individual HSs, deletions of 2 HSs, and deletion of the whole LCR and found all of the HSs had a similar spectrum of activities, albeit to different degrees. Each HS acts as an independent module to activate expression in an additive manner, and this is correlated with relocation away from the nuclear periphery. In contrast, HSs have redundant activities with respect to association with TFys and the probability that an allele is actively transcribed, as measured by primary RNA transcript FISH. The limiting effect on RNA levels occurs after ß-globin genes associate with TFys, at which time HSs contribute to the amount of RNA arising from each burst of transcription by stimulating transcriptional elongation.

A polymorphism in the TH 2 locus control region is associated with changes in DNA methylation and gene expression.

BACKGROUND: Genomewide association and epigenetic studies found a region within the RAD50 gene on chromosome 5q31 to be associated with total serum IgE levels and asthma. In mice, this region harbors a locus control region for nearby TH 2 cytokines, which is characterized by four Rad50 DNase I hypersensitive sites (RHS4-7). Among these, RHS7 seems to have the strongest impact on TH 2 differentiation. We investigated whether within the human homolog of RHS7, functional polymorphisms exist, which could affect DNA methylation or gene expression in the 5q31 locus and might have an influence on asthma status or IgE regulation. METHODS: The human RHS7 region was fine mapped using 1000 genomes database information. In silico analysis and electrophoretic mobility shift assays were used to assess SNP function. Allele-specific effects on DNA methylation were evaluated in cord blood (n = 73) and at age of 4.5 years (n = 61) by pyrosequencing. Allele-specific effects on RAD50, IL4, and IL13 expression were analyzed in 100 subjects. Associations with asthma and IgE levels were investigated in the MAGICS/ISAAC II population (n = 1145). RESULTS: Polymorphism rs2240032 in the RHS7 region is suggestive of allele-specific transcription factor binding, affects methylation of the IL13 promoter region and influences RAD50 and IL4 expression (lowest P = 0.0027). It is also associated with total serum IgE levels (P = 0.0227). CONCLUSION: A functional relevant polymorphism in the TH 2 locus control region, equivalent to RHS7 in mice, affects DNA methylation and gene expression within 5q31 and influences total serum IgE on the population level.

Active opsin loci adopt intrachromosomal loops that depend on the photoreceptor transcription factor network.

Rod and cone opsin genes are expressed in a mutually exclusive manner in their respective photoreceptor subtypes in the mammalian retina. Previous transgenic mouse studies showed that functional interactions between the distal enhancer and proximal promoter of rhodopsin and long/medium-wavelength (L/M) opsin genes are essential for regulating their cell-type-specific transcription. We have used chromosomal conformation capture assays in mouse retinas to investigate the molecular mechanism responsible for this interaction. Here we show that each opsin gene forms intrachromosomal loops in the appropriate photoreceptor subtype, while maintaining a linear configuration in other cell types where it is silent. The enhancer forms physical contacts not only with the promoter but also with the coding regions of each opsin locus. ChIP assays showed that cell-type-specific target binding by three key photoreceptor transcription factors-cone--rod homeobox (CRX), neural retina leucine zipper (NRL), and nuclear receptor subfamily 2, group E, member 3 (NR2E3)--is required for the appropriate local chromosomal organization and transcription of rod and cone opsins. Similar correlations between chromosomal loops and active transcription of opsin genes were also observed in human photoreceptors. Furthermore, quantitative chromosomal conformation capture on human retinas from two male donors showed that the L/M enhancer locus control region (LCR) loops with either the L or M promoter in a near 3:1 ratio, supporting distance-dependent competition between L and M for LCR. Altogether, our results suggest that the photoreceptor transcription factor network cooperatively regulates the chromosomal organization of target genes to precisely control photoreceptor subtype-specific gene expression.

[New data on the phylogeography and genetic diversity of the brown bear Ursus arctos Linnaeus, 1758 of northeastern Eurasia (mtDNA control region polymorphism analysis)].

An analysis of polymorphism of the fragment of the control region of mitochondrial DNA of 53 tissue samples of the brown bear Ursus arctos from several regions of the eastern part of Russia was carried out. It was found that most of the described haplotypes belong to cluster 3a, the most common in Eurasia, and do not form regionally specific haplogroups. However, among the bears from Western and Eastern Siberia, as well as the island of Kunashir, three haplotypes were identified, which are close to the haplogroup typical of Eastern Hokkaido bears. The assumption was made of the existence in Siberia and the Far East of one or more Pleistocene refugia.