Characterization of a DNA binding activity in DNAse I hypersensitive site 4 of the human globin locus control region.

A portion of the beta-globin Locus Control Region (LCR), which included DNAse I hypersensitive site 4 (HS4), was analyzed for its interactions with nuclear extracts and its contribution to LCR activity in a functional assay. In gel retardation assays, a short fragment from HS4 formed complexes with nuclear extracts from both erythroid and nonerythroid cells, and a core protected sequence 5'GACTGGC3' was revealed by DNAse I protection and methylation interference studies. This sequence resembles the binding sites of CCAAT-family members. Purified CP-2 but not CP-1 was shown to bind this HS4 sequence in a gel shift reaction, suggesting that the HS4 binding activity shares some sequence specificity with the CCAAT-factor family. Utilizing a transient expression assay in murine erythroleukemia cells, steady-state RNA levels were measured from pairs of LCR constructs linked to distinguishable beta-globin reporter genes. A short DNA fragment from HS4 which included the binding site for this novel binding activity accounted for most of the contribution to high level expression made by the entire HS4 region.     

A central role for a single c-Myb binding site in a thymic locus control region.

Locus control regions (LCRs) are powerful assemblies of cis elements that organize the actions of cell-type-specific trans-acting factors. A 2.3-kb LCR in the human adenosine deaminase (ADA) gene first intron, which controls expression in thymocytes, is composed of a 200-bp enhancer domain and extended flanking sequences that facilitate activation from within chromatin. Prior analyses have demonstrated that the enhancer contains a 28-bp core region and local adjacent augmentative cis elements. We now show that the core contains a single critical c-Myb binding site. In both transiently cotransfected human cells and stable chromatin-integrated yeast cells, c-Myb strongly transactivated reporter constructs that contained polymerized core sequences. c-Myb protein was strongly evident in T lymphoblasts in which the enhancer was active and was localized within discrete nuclear structures. Fetal murine thymus exhibited a striking concordance of endogenous c-myb expression with that of mouse ADA and human ADA LCR-directed transgene expression. Point mutation of the c-Myb site within the intact 2.3-kb LCR severely attenuated enhancer activity in transfections and LCR activity in transgenic thymocytes. Within the context of a complex enhancer and LCR, c-Myb can act as an organizer of thymocyte-specific gene expression via a single binding site.     

Functional and binding studies of HS3.2 of the beta-globin locus control region

The distal locus control region (LCR) is required for high-level expression of the complex of genes (HBBC) encoding the beta-like globins of mammals in erythroid cells. Several major DNase hypersensitive sites (HSs 1-5) mark the LCR. Sequence conservation and direct experimental evidence have implicated sequences within and between the HS cores in function of the LCR. In this report we confirm the mapping of a minor HS between HS3 and HS4, called HS3.2, and show that sequences including it increase the number of random integration sites at which a drug resistance gene is expressed. We also show that nuclear proteins including GATA1 and Oct1 bind specifically to sequences within HS3.2. However, the protein Pbx1, whose binding site is the best match to one highly conserved sequence, does not bind strongly. GATA1 and Oct1 also bind in the HS cores of the LCR and to promoters in HBBC. Their binding to this minor HS suggests that they may be used in assembly of a large complex containing multiple regulatory sequences.     

Primary structure of the goat beta-globin locus control region

The goat beta-globin cluster is composed of a triplicated four-gene set. A locus control region (LCR) containing elements homologous to 5'DNase I hypersensitive sites (HS) 1, 2, and 3 of the human beta-globin LCR has been identified at the 5' end of this locus. We determined 10.2 kb of nucleotide sequence from the goat beta-globin locus control region. Self-comparison of this sequence by dot matrix analysis revealed the presence of six complete and three incomplete artiodactyl repeats. A novel repeated element, termed D repeat, was also identified. Southern blotting analysis demonstrated that these elements exist in the goat genome as a low to medium frequency interspersed repeat family. The absence of any other large region of self-homology (direct or inverted) in the goat LCR suggests that 5'HSs 1, 2, and 3 did not arise through duplication, but rather evolved independently. By comparing goat 5'HS 1 to those of human, rabbit, and mouse, we show a greater than 80% conservation in sequence between the four species. This level of evolutionary conservation suggests that 5'HS 1 plays an important role in the regulation of beta-globin loci.     

Functional characterization of a testis-specific DNA binding activity at the H19/Igf2 imprinting control region

The DNA methylation state of the H19/Igf2 imprinting control region (ICR) is differentially set during gametogenesis. To identify factors responsible for the paternally specific DNA methylation of the ICR, germ line and somatic extracts were screened for proteins that bind to the ICR in a germ line-specific manner. A specific DNA binding activity that was restricted to the male germ line and enriched in neonatal testis was identified. Its three binding sites within the ICR are very similar to the consensus sequence for nuclear receptor extended half sites. To determine if these binding sites are required for establishment of the paternal epigenetic state, a mouse strain in which the three sites were mutated was generated. The mutated ICR was able to establish a male-specific epigenetic state in sperm that was indistinguishable from that established by the wild-type ICR, indicating that these sequences are either redundant or have no function. An analysis of the methylated state of the mutant ICR in the soma revealed no differences from the wild-type ICR but did uncover in both mutant and wild-type chromosomes a significant relaxation in the stringency of the methylated state of the paternal allele and the unmethylated state of the maternal allele in neonatal and adult tissues.     

Enhancer-blocking activity is associated with hypersensitive site V sequences in the human growth hormone locus control region

Activation of the human growth hormone gene (hGH-N) is linked to a locus control region (LCR) containing four (I-III, V) hypersensitive sites (HS). Pit-1 binding to HS I/II is required for efficient pituitary expression. However, inclusion of HS III and V, located about 28 and 32 kb upstream of the hGH-N gene, respectively, is also required for consistent hGH-N expression levels in vivo. HS V is referred to as a boundary for the hGH LCR, but no specific enhancer blocking or barrier function is reported. We examined a 547 bp fragment containing HS V sequences (nucleotides -32,718/-32,172 relative to hGH-N) for enhancer-blocking activity using a well-established transient gene transfer system and assessed these sequences for CCCTC binding factor (CTCF), which is linked to enhancer-blocking activity. The 547 bp HS V fragment decreased enhancer activity with a reverse-orientation preference when inserted between HS III enhancer sequences and a minimal thymidine kinase promoter (TKp). These sequences are associated with CTCF in human pituitary and nonpituitary chromatin. Enhancer-blocking activity with an orientation preference was further localized to a 45 bp sub-fragment, with evidence of CTCF and upstream binding factor 1 (USF1) binding; USF1 is linked more closely with barrier function. The presence of yin and yang 1 (Yy1) that cooperates with CTCF in the regulation of X-chromosome inactivation was also seen. A decrease in CTCF and Yy1 RNA levels was associated with a significant reduction in enhancer-blocking activity. Assessment of CpG-dinucleotides in the TKp indicates that the presence of HS V sequences are associated with an increased incidence of CpG-dinucleotide methylation of the GC box region. These data support association of CTCF and enhancer-blocking activity with HS V that is consistent with a role as a (LCR) boundary element and also implicates Yy1 in this process.     

Long-range looping of a locus control region drives tissue-specific chromatin packing within a multigene cluster

The relationships of higher order chromatin organization to mammalian gene expression remain incompletely defined. The human Growth Hormone (hGH) multigene cluster contains five gene paralogs. These genes are selectively activated in either the pituitary or the placenta by distinct components of a remote locus control region (LCR). Prior studies have revealed that appropriate activation of the placental genes is dependent not only on the actions of the LCR, but also on the multigene composition of the cluster itself. Here, we demonstrate that the hGH LCR ‘loops’ over a distance of 28 kb in primary placental nuclei to make specific contacts with the promoters of the two GH genes in the cluster. This long-range interaction sequesters the GH genes from the three hCS genes which co-assemble into a tightly packed ‘hCS chromatin hub’. Elimination of the long-range looping, via specific deletion of the placental LCR components, triggers a dramatic disruption of the hCS chromatin hub. These data reveal a higher-order structural pathway by which long-range looping from an LCR impacts on local chromatin architecture that is linked to tissue-specific gene regulation within a multigene cluster.     

New mutations of locus control region in Saudi sickle patients

Sickle cell anemia (SCA) is a common hematological disease affecting humans. Detection of a single base pair mutation in β-globin gene is an important diagnostic tool for SCA. The aim was to study the molecular survey of locus control regions (LCR) in Saudi patients with sickle cell anemia, and to identify the genetic variables and their clinical manifestations.MethodologyBlood samples from 69 unrelated sickle cell disease patients were obtained from the KKUH, Riyadh between 2017–2019. In this study, the DNA was extracted and PCR was performed. Additional PCR amplifications reactions covering the LCR were performed by using another different set of primers. Seven specific primer pairs were used to amplify seven regions in the locus control region (LCR) of β globin family. The generated fragments were sequenced to identify the possible alterations in this region.ResultsThe results gained from sequencing experiments revealed a wide range of genomic alterations. A total of 69 gene alterations have been recognized in the locus control region;- The first fragment LCR-HS1 shows 20 alterations; The second fragment LCR-HS2 revealed six changes; The third fragment LCR-HS3 shows many changes; The fifth LCR-HS5 region revealed four changes; The sixth fragment LCR-HS6 revealed eight changes; The seventh LCR-HS7 fragment demonstrates ten changes.ConclusionIt clear that this study has successfully identified LCR mutations for random Saudi patients with SCD. The above results should be taken further to set up management strategies to improve outcomes.