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.

Comparison of polymerases used for amplification of mitochondrial DNA from challenging hairs and hairs of various treatments.

Forensic DNA analysis of hair evidence typically involves the amplification and sequencing of the control region (CR) of the mitochondrial genome (mtgenome). In compromised hair samples, such as shed hairs, the number of mtgenome copies could be low; thus, it is imperative that the polymerase used in PCR is efficient to ensure maximum amplification. Considering this, the first phase of this study compared the yields obtained from 12 polymerases (sourced from a range of commercial companies) when amplifying the CR, hypervariable (HV) region II (HV2), and hypervariable subregion II-B (HV2B). This initial assessment was performed using mitochondrial DNA (mtDNA) extracted from 2 cm of hair adjacent to the root from three donors of different self-reported ancestries and hair color/texture. PrimeSTAR HS and KAPA HiFi HotStart consistently generated significantly higher amplicon yields (p < 0.05, ~5-fold increase) for most regions than AmpliTaq Gold DNA polymerase (the polymerase validated for use in most forensic laboratories). The second phase of this project was focused on assessing the broad utility of these top two performing polymerases for amplifying two regions of the mtgenome (CR and HV2B) from hair samples representing diverse self-reported ancestral origins (European, Latin American, African American, Asian, and Native American), characteristics/treatments (bleached, dyed, and chemically straightened), and anatomical origins (e.g., head and pubic region) (n = 41). These regions were chosen as they are the most challenging to amplify and sequence in compromised hair samples due to length (i.e., the CR is ~1.2 kb) and repeat structure (i.e., the polycytosine stretch within HV2B). The results indicated that regardless of sample type, PrimeSTAR HS and KAPA HiFi HotStart polymerases outperformed (p < 0.05) AmpliTaq Gold DNA polymerase (averaging 11- and 8-fold increased yields, respectively). The results from this study highlight that enhanced commercially available polymerases appear to significantly improve the amplification of mtDNA from challenging hair samples.

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.

OCT3/4-binding sequence-dependent maintenance of the unmethylated state of CTCF-binding sequences with DNA demethylation and suppression of de novo DNA methylation in the H19 imprinted control region.

DNA demethylation and suppression of de novo DNA methylation are activities that maintain an unmethylated state. However, the strength of these two activities at the same locus has not been estimated separately. Furthermore, the association between these two activities and the unmethylated state remains unclear. Octamer-binding transcription factor-binding sequences (OBSs) and CCCTC-binding factor-binding sequences (CBSs) within the mouse H19-imprinted control region (ICR) are involved in the induction of DNA demethylation and maintenance of the unmethylated state in mouse undifferentiated embryonic cell lines. To reveal the association between the two cis-elements and the two unmethylated state maintenance activities in maintaining the unmethylated state of the ICR, we evaluated the altered DNA methylation levels at sites that were initially methylated or unmethylated using a stable transfection-based assay, and estimated the strength of the two unmethylated state maintenance activities separately via a Poisson process model that described the DNA methylation state regulatory process. Although DNA demethylation depending on OBSs affected almost the entire ICR, DNA demethylation depending on CBSs occurred near CBSs, resulting in redundant demethylation of CBS regions. Detailed analysis of the CBS4 region suggested that OBSs were required to induce unmethylated state maintenance activities, and that CBSs-dependent activities contributed, but diminished, during incubation when protection of the CBS4 region by OBSs-dependent activities was absent. Analysis via the Poisson process model indicated that the unmethylated state at the CBS4 region was maintained by OBSs-dependent suppression of de novo DNA methylation rather than DNA demethylation. We propose that the hierarchical regulation of redundant protection of the CBS region via cooperation between the two unmethylated state maintenance activities is a potential function of the ICR that effectively maintains allele-specific methylation status in the same DNA sequence.

The role of control region mitochondrial DNA mutations in cardiovascular disease: stroke and myocardial infarction.

Recent studies associated certain type of cardiovascular disease (CVD) with specific mitochondrial DNA (mtDNA) defects, mainly driven by the central role of mitochondria in cellular metabolism. Considering the importance of the control region (CR) on the regulation of the mtDNA gene expression, the aim of the present study was to investigate the role of mtDNA CR mutations in two CVDs: stroke and myocardial infarction (MI). MtDNA CR mutations (both fixed and in heteroplasmy) were analysed in two demographically-matched case-control samples, using 154 stroke cases, 211 MI cases and their corresponding control individuals. Significant differences were found, reporting mutations m.16145 G > A and m.16311 T > C as potential genetic risk factors for stroke (conditional logistic regression: p = 0.038 and p = 0.018, respectively), whereas the m.72 T > C, m.73 A > G and m.16356 T > C mutations could act as possible beneficial genetic factors for MI (conditional logistic regression: p = 0.001, p = 0.009 and p = 0.016, respectively). Furthermore, our findings also showed a high percentage of point heteroplasmy in MI controls (logistic regression: p = 0.046; OR = 0.209, 95% CI [0.045-0.972]). These results demonstrate the possible role of mtDNA mutations in the CR on the pathogenesis of stroke and MI, and show the importance of including this regulatory region in genetic association studies.

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.

Variations in mitochondrial DNA control region among Malay and Chinese subpopulations (sequence 16000-16200).

DNA variations are alterations found in DNA sequence, occurring in both nuclear DNA and mitochondrial DNA. Variations might differ in individual following population, respectively. The aim of this study was to find variations in target sequence of mtDNA (16000-16200) to be used as marker in Malay and Chinese population. A total of 30 buccal swab samples from 20 Malay and 10 Chinese subjects were collected and preserved on FTA card. The FTA card that contained DNA sample was punched to be included into polymerase chain reaction mixture. Amplification was carried out and the products were sequenced. Sequence variations were found in both Malay and Chinese populations. A total of nine variations (16129, 16108, 16162, 16172, 16148, 16127, 16173, 16099 and 16100) were found in Malay population while a total of seven variations (16129, 16104, 16111, 16109, 16164, 16170 and 16136) were found in Chinese population. Nucleotide position 16129 was found as variation in both Malay and Chinese populations. This study implies that np 16129 can be used as a marker for Malaysian population. For further investigation, the length of the target sequence may be increased to obtain more variations that can be used as markers. This will increase the discrimination power of Malaysian population.

[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.

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.

A Systematic Bioinformatics Approach to Motif-Based Analysis of Human Locus Control Regions.

Locus control regions (LCRs), cis-acting, noncoding regulatory elements with strong transcription-enhancing activity, are conserved in sequence and organization, and exhibit strict gene-specific expression. LCRs have been reported and studied in several mammalian gene systems, signifying that they play an important role in eukaryotic gene expression control. Their highly regulated, stable, and precise levels of expression have made them a strong candidate for use in gene therapy vectors. In this study, we attempted to determine the unique signatures of human LCRs by analyzing a data set of LCR sequences for the presence of motifs through systematic bioinformatics approach. Using web-based regulatory sequence analysis tools (RSAT), motif-based analysis was performed. Detected significant motifs were analyzed further for their identity using Tomtom tool. RSAT analysis revealed that significant motifs are existent within the LCRs. Identity analysis using Tomtom showed that detected significant motifs were comparable with known transcription factor (TF) binding sites and the top scoring motifs belong to zinc finger-containing proteins, an important group of proteins involved in a variety of cellular activities. Correspondence to segment of known motif indicates the biological relevance of the detected motifs. Motif-based analysis is valuable for analyzing the various characteristics of sequences, notably TF binding models in this study. Owning to their unique expression control abilities, LCRs form an important component of integrating vectors, therefore identification of unique signatures present within LCR sequences will be instrumental in the design of new generation of regulatory elements containing LCR sequences.

Population structuring of Channa striata from Indian waters using control region of mtDNA.

Striped snakehead (Channa striata) is a freshwater species of early Miocene period belonging to family Channidae. The genetic variability of the snakehead populations in India was not well known. Present study was undertaken using 149 sequences of control region of mitochondrial DNA from seven geographically distinct populations of Indian water, which resulted in 46 haplotypes with 137 variable nucleotide sites (60 singletons and 77 parsimony informative) and the nucleotide frequencies was: A = 33.0, T = 28.1, G = 15.4, and C = 23.5%. The presence of low-frequency of younger haplotypes with a large number of singletons indicates the absence of dominant haplotype. Hierarchical AMOVA showed highly significant genetic differentiation (FST = 0.56, p < .00) among the populations. The pattern of genetic differentiation was not consistent with geographical distributions. AMOVA identified three genetically heterogeneous clades. The significant finding of Imphal and Chaliyar was that they were found genetically close to each other, but geographically much isolated from each other. The inference of the study suggests that the hypothesis of the single panmictic population of C. striata in Indian waters is rejected.

Transvection-like interchromosomal interaction is not observed at the transcriptional level when tested in the Rosa26 locus in mouse.

Long-range associations between enhancers and their target gene promoters have been shown to play critical roles in executing genome function. Recent variations of chromosome capture technology have revealed a comprehensive view of intra- and interchromosomal contacts between specific genomic sites. The locus control region of the ß-globin genes (ß-LCR) is a super-enhancer that is capable of activating all of the ß-like globin genes within the locus in cis through physical interaction by forming DNA loops. CTCF helps to mediate loop formation between LCR-HS5 and 3'HS1 in the human ß-globin locus, in this way thought to contribute to the formation of a "chromatin hub". The ß-globin locus is also in close physical proximity to other erythrocyte-specific genes located long distances away on the same chromosome. In this case, erythrocyte-specific genes gather together at a shared "transcription factory" for co-transcription. Theoretically, enhancers could also activate target gene promoters at the identical loci, yet on different chromosomes in trans, a phenomenon originally described as transvection in Drosophilla. Although close physical proximity has been reported for the ß-LCR and the ß-like globin genes when integrated at the mouse homologous loci in trans, their structural and functional interactions were found to be rare, possibly because of a lack of suitable regulatory elements that might facilitate such trans interactions. Therefore, we re-evaluated presumptive transvection-like enhancer-promoter communication by introducing CTCF binding sites and erythrocyte-specific transcription units into both LCR-enhancer and ß-promoter alleles, each inserted into the mouse ROSA26 locus on separate chromosomes. Following cross-mating of mice to place the two mutant loci at the identical chromosomal position and into active chromation in trans, their transcriptional output was evaluated. The results demonstrate that there was no significant functional association between the LCR and the ß-globin gene in trans even in this idealized experimental context.

Comparative analysis of the Liriomyza chinensis mitochondrial genome with other Agromyzids reveals conserved genome features.

Liriomyza chinensis is a serious pest of onions in many countries, especially in East Asia. We sequenced the complete mitochondrial genome of this species and compared it with five other Agromyzidae species. The L. chinensis mitogenome is a double-stranded 16,175 bp circular molecule with an A + T content of 78.3%. It contains 37 genes and a control region as do the sequenced Liriomyza species. The mitogenomes of L. chinensis and other Agromyzidae species showed a clear bias in nucleotide composition with a positive AT-skew. Most PCGs used standard ATN as start codons, and TAN as termination codons. The tRNAs exhibited the typical clover-leaf structure, except for tRNASer(AGN) and the two rRNA genes are conserved with those of other Agromyzids. The L. chinensis mitogenome control region included several conserved regions, including a poly-T, two (TA)n and one poly-A stretch, which are considered important replication and transcription. The 13 PCGs were used to study the phylogeny of L. chinensis and five related Agromyzids. Analysis by maximum likelihood, Bayesian inference and genetic distance suggest congruent phylogenetic relationships in Liriomyza spp. and provide a useful supplement to taxonomic classification by morphology.

mtDNA sequence diversity of Hazara ethnic group from Pakistan.

The present study was undertaken to investigate mitochondrial DNA (mtDNA) control region sequences of Hazaras from Pakistan, so as to generate mtDNA reference database for forensic casework in Pakistan and to analyze phylogenetic relationship of this particular ethnic group with geographically proximal populations. Complete mtDNA control region (nt 16024-576) sequences were generated through Sanger Sequencing for 319 Hazara individuals from Quetta, Baluchistan. The population sample set showed a total of 189 distinct haplotypes, belonging mainly to West Eurasian (51.72%), East & Southeast Asian (29.78%) and South Asian (18.50%) haplogroups. Compared with other populations from Pakistan, the Hazara population had a relatively high haplotype diversity (0.9945) and a lower random match probability (0.0085). The dataset has been incorporated into EMPOP database under accession number EMP00680. The data herein comprises the largest, and likely most thoroughly examined, control region mtDNA dataset from Hazaras of Pakistan.

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.

Erythroid specific activator GATA-1-dependent interactions between CTCF sites around the ß-globin locus.

CTCF sites (binding motifs for CCCTC-binding factor, an insulator protein) are located considerable distances apart on genomes but are closely positioned in organized chromatin. The close positioning of CTCF sites is often cell type or tissue specific. Here we analyzed chromatin organization in eight CTCF sites around the ß-globin locus by 3C assay and explored the roles of erythroid specific transcription activator GATA-1 and KLF1 in it. It was found five CTCF sites convergent to the locus interact with each other in erythroid K562 cells but not in non-erythroid 293 cells. The interaction was decreased by depletion of GATA-1 or KLF1. It accompanied reductions of CTCF and Rad21 occupancies and loss of active chromatin structure at the CTCF sites. Furthermore Rad21 occupancy was reduced in the ß-globin locus control region (LCR) hypersensitive sites (HSs) by the depletion of GATA-1 or KLF1. The role of GATA-1 in interaction between CTCF sites was revealed by its ectopic expression in 293 cells and by deletion of a GATA-1 site in the LCR HS2. These findings indicate that erythroid specific activator GATA-1 acts at CTCF sites around the ß-globin locus to establish tissue-specific chromatin organization.

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.