Trans-acting factors from the human fetal liver bindingto the human ε-globin gene silencer

The developmental stage-specific silencing of the human ε-globin gene during embryonic life is controlled, inpart, by the silencer (-392bp～-177bp) upstream of thisgene. In order to elucidate its role, the nuclear extractfrom the human fetal liver has been prepared and the interactions between trans-acting factors and this silencerelement have been examined. By using DNasel footprinting assay, a major protected region from -278bp to -235bpwithin this silencer element was identified. Furthermore,we found in gel mobility shift assay and Southwestern blotting assay that there were at least four trans-acting factors (MW ≈ 32, 28, 26 and 22kD) in the nuclear extractisolated from the human fetal liver, which could specifically bind to this region. Our results suggested that thesetrans-acting factors might play an important role in silencing the human embryonic ε-globin gene expression at thefetal stage through the interactions with this silencer.

Epidemiological Survey of Hemoglobinopathies Based on Next-Generation Sequencing Platform in Hunan Province,China

Objective This study was aimed at investigating the carrier rate of,and molecular variation in,α-andβ-globin gene mutations in Hunan Province.Methods We recruited 25,946 individuals attending premarital screening from 42 districts and counties in all 14 cities of Hunan Province.Hematological screening was performed,and molecular parameters were assessed.Results The overall carrier rate of thalassemia was 7.1%,including 4.83%forα-thalassemia,2.15%forβ-thalassemia,and 0.12%for bothα-andβ-thalassemia.The highest carrier rate of thalassemia was in Yongzhou(14.57%).The most abundant genotype ofα-thalassemia andβ-thalassemia was-α^(3.7)/αα(50.23%)andβ^(IVS-Ⅱ-654)/β^(N)(28.23%),respectively.Fourα-globin mutations[CD108(ACC>AAC),CAP+29(G>C),Hb Agrinio and Hb Cervantes]and sixβ-globin mutations[CAP+8(C>T),IVS-Ⅱ-848(C>T),-56(G>C),beta nt-77(G>C),codon 20/21(-TGGA)and Hb Knossos]had not previously been identified in China.Furthermore,this study provides the first report of the carrier rates of abnormal hemoglobin variants andα-globin triplication in Hunan Province,which were 0.49%and 1.99%,respectively.Conclusion Our study demonstrates the high complexity and diversity of thalassemia gene mutations in the Hunan population.The results should facilitate genetic counselling and the prevention of severe thalassemia in this region.

Studies on DNA-protein interactions in the upstream regulatory region of the human ε-globin gene promoter

The erythroid- and developmental stage-specific expression of the human ε-globin gene is controlled, in part,by the 5’-flanking DNA sequence of this gene. In the present study, we have used DNA-protein binding assays to identify trans-acting factors which regulate the temporal expression of the human ε-globin gene during development. Using gel mobility shift assays and DNasel footprinting assays, a nuclear protein factor (termed ε-SSF1) in the nuclear extracts from mouse haematopoietic tissues at d 11 and d 13 of gestation was identified. It could specifically bind to the positive control region (between -535 and -453bp) of the human ε-globin gene. We speculated that the E-SSF1 might be an erythroid- and developmental stage-specific activator. In addition, we found another nuclear protein factor (termed ε-R1) in the nuclear extract from mouse fetal liver at d 18 of gestation, which could strongly bind to the silencer region (between -392 and -177bp) of this gene. Therefore, we speculated that the ε-R1 might be an erythroid- and developmental stagespecific repressor. Our data suggest that both ε-SSF1 and ε-R1 might play important roles in developmental regulation of the human ε-globin gene expression during the early embryonic life. On the other hand, we observed that the binding patterns of nuclear proteins from three cell lines (K562, HEL and Raji) to these regulatory regions were partially different. These results suggest that different trans-acting factors in K562, HEL and Raji cells might be responsible for activating or silencing the human ε-globin gene in three different cell lines.

A DNA-binding protein factor in K562 nuclear extract interacts with positive control region (PCR) in the 5'flanking sequence of human β-globin gene

It has been known that there are at least three regulatory regions (NCR1, NCR2 and PCR) in the 5'-flanking sequence (from -610 bp to +1bp) of human β-globin gene and that the function of PCR is unique to the human erythroleukemia (K562) cells. Here we have detected a DNA-binding protein factor (termed NFEa) in K562 cells, which can bind specifically to the PCR of human β-globin gene. The sequence of the binding site is 5'ACTGATG3' (between -222 bp and -216 bp). The NFEa is erythroid-specific and perhaps specific for K562 cells. It seemed that this factor differed from the erythroid-specific tran-scriptional factor (NFE-1) using competition assay. The presence of the NFEa further supported that the function of the cis-acting element PCR was specific for K562 cells, and helps us to understand the mechanism of the regulation of the expression of human β-globin gene in the human K562 cells.

The 5'-flanking cis-acting elements of the human ε-globin gene associates with the nuclear matrix and binds to the nuclear matrix proteins

The nucleax mains attachment regions(MARs) and the binding nuclear matrix proteins in the 5’-flalildng cisacting elements of the humanε-globin gene have been examined. Using in vitro DNA-matrix binding assay,it has been shown that the positive stage-specific regulatory element (ε-PREII, -446bp-419bp) upstream of this gene could specifically associate with the nuclear matrix from K562 cells, indicating thatε-PREII mad be an erythroidspecilic facultstive MAR. In gel mobility shift assay and Southwestern blotting assal an eothroid-specific nuclear matrix protein (ε-NMPk) in K562 cells has been revealed to bind to this positive regulatory element (E-PREII). Furthermore, we demonstrated that the silencer (-392hp -177bp) uP8tream of the humanε-globin gene could associate with the nuclear matrices from K562, HEL and Raji cells. In addition, the nucleax matrix proteins prepared from these three cell lines could also bind to this silencer, suggesting that this silencer element linght be a constitutive nuclear mains attachment region (constitutive MAR). Our results demonstrated that the nucleax madrid and nuclear mains proteins lxilght play an important role in the regulation of the human 5-globin gene expression.

Proteins binding to the 5'-flanking regulatory elements of the human β-globin gene

The binding of nuclear proteins prepared from mouse erythroid tissue in different developmental stages to the 5'-flanking regulatory elements of human β-globin gene, two negative control regions(NCR1,-610 to -490 bp;NCR2, -338 to-233bp), was identified.Two stage specific protein factors corresponding to embryonic and fetal stages were found to be capable of binding to NCR2.These data provided evidence that the cis acting elements of the 5'-flanking region might be involved in the developmental control of β-globin gene and NCR2 might be responsible in part for the silence of β-globin gene in the embryonic and fetal stages.

Identification of the development stage-specific factors in mouse fetal liver binding to the human β-globin gene promoter

In order to elucidate the molecular mechanisms of globin gene expression during embryonic development, the nuclear extracts from mouse hematopoietic tissue at different stages of development have been prepared. By using DNase I footprinting and gel mobility shift assays, the binding of protein factors in these extracts to the human βglobin promoter was analyzed. The differences in the binding patterns of protein factors during development were observed. An erythroid-specific and stage-specific nuclear protein in the nuclear extract from d 18 mouse fetal liver was identified, which can bind to the sequence (from -66bp to -90bp) of human β-globin promoter. We therefore speculate that the function of this cis-acting element may be similar to stage selector element (SSE) in chieken βA- promoter.

Co-Inheritance of Beta &Delta-Globin Gene (HbYialousa) Mutations in an Iranian <i>β</i>-Thalassemia Carrier

Introduction: Beta-thalassemia is characterized by absence or reduced synthesis of the β-globin. Carriers of β-thalas- semia, typically have microcytic hypochromic anemia and elevated hemoglobin HbA2 and normal HbF level. On the other hand carriers of severe alpha-thalassemia also have similar CBC parameters to that of β-thalassemia with normal HbA2 level. Co-presence of mutations in the β-globin and delta-globin genes (point mutations or deletions) usually give normal HbA2 and elevated HbF level. We report a β-thal carrier with normal level of HbA2 and increased level of HbF who had a point mutation in CD39 on the beta-globin gene and a point mutation in CD27 on the δ-globin gene named Hb-Yialousa. Materials & Methods: An individual with low hematological indices, normal HbA2 and elevated HbF was referred to our center as routine premarital screening program. Mutations in the β-globin and δ-globin genes were screened using ARMS and sequencing methods. Results: The mutation in β- and δ-globin genes were identified as CD39 and CD27 (HbYialousa) respectively. No point mutation or deletion in α-globin gene was identified. Discussion: We showed that normal HBA2 with elevated HbF level is due to co-inheritance of delta-globin gene mutation with mutation in the β-globin gene. When screening for β-thalassemia, one has to either rule out presence of α-globin gene mutation of mutation in the delta-globin gene.

Identification of a NF-кB site in the negative regulatory element (ε-NRAII) of human ε-globin gene and its binding protein NF-кB p50 in the nuclei of K562 cells

The developmental control of the human e-globin gene expression is mediated by transcription regulatory elements in the 5’ flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-кB consensus sequence resides in the negative regulatory element (-3028bp～ -2902bp, termed ε-NRAII) 5’ to the cap site of this gene. NRF DNA fragment (-3010bp～ -2986bp) containing the NF-кB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-кB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-кB motif) could not, suggesting that the binding protein is a member of NF-кB/Rel family. Western blot assay demonstrated that the molecular weight of NF-кB protein in the nuclei of K562 cells is 50ku. We suggested that NF-кB p50 may play an important role in the regulation of human c-globin gene expression.

Identification of a NF-кB site in the negative regulatory element (ε-NRAII) of human ε-globin gene and its binding protein NF-кB p50 in the nuclei of K562 cells

The developmental control of the human e-globin gene expression is mediated by transcription regulatory elements in the 5' flanking DNA of this gene. Sequence analysis has revealed a DNA motif (GGGGAATTTGCT) similar to NF-кB consensus sequence resides in the negative regulatory element (-3028bp～ -2902bp, termed ε-NRAII) 5' to the cap site of this gene. NRF DNA fragment (-3010bp～ -2986bp) containing the NF-кB motif similar sequence was synthesized and used in electrophoresis mobility shift assay (EMSA) and competitive analysis. Data showed that a protein factor from nuclear extracts of K562 cells specifically interacted with NRF DNA fragment. The synthetic NF DNA fragment (containing NF-кB consensus sequence) could competed for the protein binding, but MNF DNA fragment (mutated NF-кB motif) could not, suggesting that the binding protein is a member of NF-кB/Rel family. Western blot assay demonstrated that the molecular weight of NF-кB protein in the nuclei of K562 cells is 50ku. We suggested that NF-кB p50 may play an important role in the regulation of human c-globin gene expression.

Screening and functional analysis of the long-range interaction elements ofβ-globin genes

Objective:Studies have shown thatβ-globin gene presents a selective expression transformation mechanism during development,and its upstream locus control region(LCR)regulates the expression pattern ofβ-globin gene family.To further explore the molecular network ofβ-globin gene expression regulation,other long-range regulatory elements that may be involved in the regulation ofβ-globin gene expression were screened and the dynamic regulation and transformation mechanism ofβ-globin gene was deeply studied.Methods:Promyelocytic cells were induced to differentiate by all-trans retinoic acid.β-globin gene promoter region and LCR were used as the target sites for circular chromosome conformational capture(4C)analysis.Through sequencing and regulatory element analysis,the sites interacting withβ-globin family loci were screened in the whole genome.Results:According to the results of 4C sequencing,the sites that interact with HBD promoter region and LCR were screened.Verified by chromosome conformational capture(3C),the results were consistent with those of sequencing.The functional analysis of regulatory elements by formaldehyde-assisted separation regulatory elements and Epiregio online website showed that the screening sites AC105129.4,AL354707.17,AC078785.22 and AC021646.35 were all potential regulatory elements involved inβ-globin gene.Conclusion:The interaction between 4C screening site and anchor site showed the complex spatial organization ofβ-globin family loci in the nucleus.

1例中间型β-地中海贫血的临床诊断和基因检测

目的通过回顾性分析1例中间型β-地中海贫血(地贫)的诊断及基因检测流程,总结中间型地贫的诊断策略。方法分析1例β-地贫合并α-地贫基因型患儿的临床表现,通过地贫基因多重PCR检测及DNA序列测定明确诊断,结合相关文献复习,探讨影响β-地贫表现型的基因因素。结果该地贫患儿基因型为β珠蛋白基因41/42突变的杂合子合并αααanti3.7基因杂合子。结论中间型地贫的诊断,需要临床表现与基因病变相一致,多重PCR结合DNA序列测定等方法可确诊罕见型基因突变。

HKαα及其复合-α4.2缺失型α地中海贫血的临床分析

目的:探讨香港型地中海贫血(HKαα)及其复合-α^(4.2)缺失型α地中海贫血(HKαα/-α^(4.2))的临床表型和血液学特征,为临床诊疗和遗传咨询提供参考。方法:收集2021年5月至2022年3月在广西医科大学第一附属医院进行地中海贫血检测的病例,采用血细胞分析仪进行血液学分析,高效液相色谱法(HPLC)进行血红蛋白分析;采用跨越断裂点聚合酶链反应(Gαp-PCR)和荧光PCR熔解曲线法(FCMA)进行α和β地中海贫血基因分析。结果:共检出165例患者,其中-α^(3.7)或-α^(4.2)缺失型α地中海贫血杂合子90例,--^(SEA)/-α^(3.7)或--^(SEA)/-α^(4.2)缺失型血红蛋白H(Hb H)病75例。在165例中检出1例罕见的HKαα/-α^(4.2)及5例HKαα。基因型为HKαα/-α^(4.2)的患者血常规检测结果:红细胞计数(RBC)5.59×10^(12)/L,血红蛋白(Hb)166 g/L,红细胞平均容积(MCV)88.60 fL,红细胞平均血红蛋白量(MCH)29.70 pg,红细胞平均血红蛋白浓度(MCHC)335.00 g/L;5例HKαα的患者血常规结果:RBC(4.77±0.26)×10^(12)/L,Hb(135.68±4.37)g/L,MCV(86.25±4.05)fL,MCH(28.47±1.12)pg,MCHC(330.48±3.75)g/L。6例患者中,Hb分析结果均未见异常,均无贫血表现,无黄疸及肝脾肿大。基因分析结果:6例患者ααα^(αnti4.2)基因检测结果均为阳性,1例基因型为HKαα/-α^(4.2),5例基因型为HKαα。结论:首次发现HKαα/-α^(4.2)患者无贫血症状,且血液学检测正常,提示此类病例在临床上较容易漏诊和误诊。

Developmental stage-specific factors in the mouse haematopoietic tissues binding to the 5'-flanking as-acting elements of humanε-globin gene

The human ε-globin gene is expressed in a tissue-specific and developmental stage-specific manner. During the earliest stage of gestation, this gene is expressed in the yolksac, but is silenced completely at the 6th-8th weeks of gestation. Recently, several studieson the transgenic mice have shown that the 5′-flanking DNA sequences of human

β地中海贫血合并α珠蛋白基因三联体2例临床分析

目的探讨β地中海贫血(地贫)合并α珠蛋白基因三联体致中间型地贫的诊断。方法回顾分析2例β杂合子患儿的临床资料,以及患儿外周血β珠蛋白基因测序及α珠蛋白基因三联体检测结果。结果例1,女,4岁,为β^(CD41-42)杂合子;例2,男,13岁,为β^(CD17)杂合子。2例患儿的临床表现均为中重度贫血、肝脾肿大,β珠蛋白基因测序未发现罕见变异点。例1的Gap-PCR特异性引物检测ααα^(anti4.2)片段阳性,例2荧光定量PCR相对定量检测ααα^(anti3.7)片段阳性。结论β杂合子且未见罕见变异,但临床表现为中重度贫血时,应考虑存在α珠蛋白基因三联体。

The in vitro reconstitution of nucleosome and its binding patterns with HMG1/2 and HMG14/17 proteins

Using atomic force microscopy (AFM), the dynamic process of the in vitro nucleosome reconstitution followed by slow dilution from high salt to low salt was visualized. Data showed that the histone octamers were dissociated from DNA at 1M NaCl. When the salt concentration was slowly reduced to 650 mMand 300 mM, the core histones bound to the naked DNA gradually. Once the salt concentration was reduced to 50 mM the classic 'beads-on-a-string' structure was clearly visualized. Furthermore, using the technique of the in vitro reconstitution ofnucleosome,the mono- and di- nucleosomes were assembled in vitro with both HS2core (-10681 to -10970 bp) and NCR2 (-372to -194 bp) DNA sequences in the 5'flanking sequence of human b-globin gene. Data revealed that HMG 1/2 and HMG 14/17 proteins binding to both DNA sequences are changeable following the assembly and disassembly of nucleosomes. We suggest that the changeable binding patterns of HMG 14/17 and HMG1/2 proteins with these regulatory elements may be critical in the process of nucleosome assembly, recruitment of chromatin-modifying activities, and the regulation of human b-globin gene expression.

Trans-acting factor binding to negative control region (NCR2) in 5' flanking sequence of human β-globin gene

Human β-globin gene family provides an ideal model for studying the expression and regulation of eukaryotic gene. The five transcriptional active genes arranged 5′ε-~Gγ-~Aγ-δ-β 3′ in the order that they are expressed during development. However, the molecular regulatory mechanism of the human globin gene expression remains to be defined. β-globin gene normally expressed in the adult bone marrow, but not in the embryonic stage, which

STM Studies on Tertiary Structure of a Negative Control Region (NCR1) of Human β-globin Gene

One of the crucial tasks of fundamental studies on modern biology is to explore the regulatory mechanisms of gene expression. Yet so far little has been known about the fine structural changes induced by the interaction between the DNA and the proteins. The major obstacles arise from the fact that it is not easy to crystallize the protein-DNA complex that is generally small in amount. This prevents researchers from gaining knowledge of the local structure with X-ray crystallography. On the other hand, the resolution of the electron microscope is not high enough to reveal structural details in nanometer scale.

Interaction Between HMG Proteins (1+2) and the Negative Regulatory Region 1(NCR1) in the 5'-flanking Sequence of the Human β-globin Gene

The pattern of high mobility group proteins 1 and 2 (HMG1,2) interaction with the 5’-flanking sequence of the human β-globin gene has been analyzed by scanning tunnelling microscopy (STM). A 200 bp negative regulatory region in the 5’-flanking sequence of the human β-globin gene can be folded by HMG proteins 1 and 2 into a circular structure (diameter 70±6) with a linear tail which seems to be a left-handed double helix structure.

Binding of HMG Proteins to the 5'_Flanking Sequence of Human β-Globin Gene

Our previous studies have identified that there are at least three regulatory regions (two negative regions and one positive region) in the 5'-flanking sequence of human β-globin gene (-610 to +1 bp). The binding of HMG proteins to both negative regulatory regions was examined by the gel mobility shift and DNase I protection assays.In gel mobility shift assay,we observed that HMG proteins 1 and 2 could bind to both negative regulatory regions (NCR1 and NCR2).Using the gel shift competition assay,we identified that the binding proteins between the two regions are different from each other.DNase I protection analysis shows that HMG proteins 1 and 2 only bind to one site (between-560 and-533 bp) in NCR1.However,two protected regions can be detected in NCR2, one between-272 and-252 bp relative to the cap site, the other between-306 and-329 bp.We also observed that HMG proteins 14 and 17 could not bind to both negative regions, so it seems that HMG proteins 1 and 2 may play an important role in the regulation of β-globin expression through DNA-protein interaction or through protein-protein interaction.