XIAO Yi-shu, XU Lan, LIU Chun-ya, DU Le, SUN Yuan-tian, LI Dong-na, REN Li-cheng

Department of Biology, Hainan Medical University, Haikou 571199, China

Keywords:

ABSTRACT Objective: Studies have shown that β-globin gene presents a selective expression transformation mechanism during development, and its upstream locus control region (LCR)regulates the expression pattern of β-globin gene family.To further explore the molecular network of β-globin gene expression regulation, other long-range regulatory elements that may be involved in the regulation of β-globin gene expression were screened and the dynamic regulation and transformation mechanism of β-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 with β-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 in β-globin gene.Conclusion: The interaction between 4C screening site and anchor site showed the complex spatial organization of β-globin family loci in the nucleus.

1.Introduction

The clustered distribution of eukaryotic genes in the nucleus is a common phenomenon, and these clustered genes show cooperative expression regulation [1-3].The understanding of the mechanism of gene expression regulation by chromosome conformation capture(3C) technology has changed from the traditional linear regulation model to the three-dimensional spatial regulation model.Circular chromosome conformational capture (4C) technique is used to analyze gene loci interacting with specific sites in the whole genome.the key of this technique is that the known DNA fragments(anchor) are linked to unknown DNA fragments.Through reverse PCR amplification and sequence analysis of PCR products, the location of chromatin interaction and the possibility of chromatin interaction can be determined.This technique can be combined with high-throughput sequencing analysis to capture all gene loci interacting with 4C anchor sites in the whole genome [4-6].

The β-globin gene cluster is located on chromosome 11 and contains globin gene (HBB) and four other β-globin-like genes:-globin gene expressed in embryo (HBE), γ-globin gene expressed in fetal stage (HBG1/HBG2) and δ-globin gene expressed in adulthood (HBD).The expression and regulation of these genes are precise and have a high developmental sequence, which is an example for the study of tissue specificity and developmental regulation of transcription [7,8].The locus control region (Locus control regions, LCR) located in the upstream 6～20 kb region of β-globin gene plays an important role in the regulation of globin gene expression in the whole gene cluster [9,10].

In order to further study the molecular network of β-globin family gene expression regulation, 4C technique was used to capture the sites that potentially interact with β-globin family loci throughout the genome.3C technique was used to further analyze the interaction frequency between potential interaction sites [11-14].Formaldehydeassisted isolation regulatory elements (FAIRE) were used to analyze the openness of these sites [15].Furthermore, the functional prediction and analysis of regulatory elements are carried out by Epiregio online tool [16].In this paper, we will analyze the molecular mechanism of dynamic regulation of β-globin gene.

2.Materials and methods

2.1 Materials

Promyelocytic leukemia cells (NB4) are preserved in our laboratory.Cell liquid medium DMEM and fetal bovine serum were purchased from Meilune.All-trans retinoic acid (ATRA) was purchased from Sigma.T4 DNA ligase, DNA restriction endonuclease Dpn II and Hind III are NEB products.Protein A/G Plus- Agarose and anti-CTCF are Upstate products.DNA purification kit is TIANGEN product.The TB Green TM Premix DimerEraserTMused in DNA Marker, ExTaq enzyme and quantitative PCR are Takara Bio products.The trace PCR product recovery kit is Qiagen product.

2.2 Cross-linked nuclear preparation

Promyelocytic cells were collected after treatment with 20 μmol/L ATRA for 72 h.The preparation of cross-linked nuclei was mainly carried out according to the methods reported in the literature [14,17-19].1% formaldehyde fixed at room temperature for 10 minutes,0.125 mol/L glycine fixed 5 min to quench formaldehyde, cells were collected and cracked on ice for 1.5 hours, and some samples were taken to count the number of cells in the sample.

2.3 Preparation of 4C sample

4C sample preparation methods mainly refer to the methods reported in the literature [4,14,18].The cross-linked nuclei were taken and digested overnight by restriction endonuclease Dpn II 37 ℃.After inactivation of Dpn II, the digested products were ligated at 16℃.4C sample DNA recovery was extracted by phenol-chloroform,and the 4C sample was quantified after purification.

2.4 Optimization of 4C nested reverse PCR amplification conditions

4C PCR amplification condition optimization reference reporting method [14].The annealing temperatures of 56 ℃, 58 ℃ and 60 ℃ were selected to simulate the 4C scheme to analyze the annealing temperature of PCR.Secondly, the samples with template concentration of 100 ng and 200 ng were amplified by PCR, and the sensitivity and diversity of primers were analyzed.The PCR amplification method is based on the product description of Taq enzyme ExTaqTM provided by Baori Medical Biology Co., Ltd.

2.5 Chromatin immunoprecipitation (ChIP) analysis

Anti-CTCF chromatin immunoprecipitation (anti-CTCF ChIP)was carried out mainly with reference to the methods reported in the literature and the instructions of the kit [18-20].First, the chromatin of the cross-linked nucleus was broken into fragments by ultrasound.Add anti-CTCF serum and shake at 4 ℃ overnight; add Protein A/G Agarose, 4 ℃ concussion overnight.Elution Buffer and protease K were added to decross-link at 65 ℃.DNA samples were recovered by trace PCR recovery kit.The enrichment efficiency of ChIP samples was analyzed by quantitative PCR.

2.6 3C analysis

3C sample preparation method, the method reported in reference[17-19].The cross-linked nuclei were digested by restriction endonuclease Hind III.After inactivation of Hind III, the enzyme digested product was linked to the sample at 16 ℃.The DNA recovery of 3C samples was extracted and purified by phenolchloroform.The 3C samples were quantified by quantitative PCR.The PCR amplification method was carried out according to TB GreenTMPremix DimerEraserTMproduct instructions.

2.7 FAIRE analysis

FAIRE sample preparation methods mainly refer to the methods reported in the literature [21].The cross-linked nuclei were extracted by phenol-chloroform, the upper aqueous phase DNA samples were recovered and purified, and the FAIRE samples were obtained.FAIRE samples were quantified and quantitative PCR was used for quantitative analysis.

2.8 Functional analysis of potential remote interaction elements of β-globin gene

The 4C sequencing data were compared with the human genome sequence by BLAST 2.10.1+ software, and the functional prediction of regulatory elements was carried out by Epiregio [16] online tool.

2.9 Statistical processing

Statistical analysis by GraphPad Prism 8 software, P＜0.05 was considered to be statistically significant.

3.Results

3.1 Analysis of restriction endonuclease digestion efficiency of cross-linked nuclei by Dpn II

In the process of preparing 4C samples, it is necessary to analyze the efficiency of cross-linked nuclei digested by Dpn II.Genomic DNA is a control system without Dpn II enzyme, and the operation flow is the same as that of 4C preparation.The purpose is to observe whether there is DNA degradation during restriction endonuclease digestion.10% of the cross-linked cells digested by restriction endonuclease were checked for restriction efficiency analysis,and the results of gel electrophoresis were shown in Figure 1.The genomic DNA is a complete DNA, indicating that the sample is not degraded.The DNA bands before induction (Uninduced) and after induction (ATRA-induced) were diffuse, which met the requirements of the follow-up experiment.It is proved that Dpn II has high digestion efficiency.

Fig 1 Analysis of enzyme digestion efficiency by gel electrophoresis

3.2 Optimization of 4C PCR Amplification conditions

4C technology enables cross-linking between different gene loci close to each other and being linked together to form circular bimolecules [4].Most of the 4C sample DNA are linear molecules that do not form a ring, and a few are intended ring molecules.Two rounds of nested PCR are needed to screen the regulatory elements by PCR, so it is necessary to optimize the PCR program in the process of 4C sample amplification to make the PCR system sensitive and diverse.Firstly, the annealing temperature of β-globin family gene loci (LCR/HBB/HBD/HBE/HBG1/HBG2) primers was analyzed by simulating 4C scheme.The result is shown in figure 2.The bands from left to right are 56 ℃, 58 ℃ and 60 ℃, respectively.Through the results of agarose gel electrophoresis, it can be observed that the samples can be effectively amplified at three different annealing temperatures, and all of them are the desired dispersion bands, indicating that the amplified samples are diverse.In order to reduce the non-specific amplification, 60 ℃ was selected as the annealing temperature for 4C PCR amplification.

Fig 2 Analysis of different annealing temperatures by gel electrophoresis

Sensitivity analysis of β-globin family loci (LCR/HBB/HBD/HBE/HBG1/HBG2) PCR amplification.As shown in figure 3, agarose gel electrophoresis shows that the templates of 100 ng and 200 ng can be effectively amplified by two rounds of PCR amplification, and after two rounds of amplification, the bands tend to disperse, indicating that the amplification is diverse.

Fig 3 Analysis of 4C amplification sensitivity

3.3 Capture of interacting loci in 4C samples by reverse nested PCR

In order to study the chromosome interaction network of β-globin loci, LCR and Dpn II digested DNA fragments containing the promoter region of β-globin related loci were selected as 4C anchoring fragments.According to the optimized procedure, the nested reverse PCR amplification conditions are as follows: 96℃ 30 s, 60 ℃ 30 s, 68 ℃ 2 min, a total of 30 cycles.Agarose gel electrophoresis analysis of PCR amplification products met the requirements of follow-up experiments.The PCR products were purified by ultrasound and sent to the sequencing company for sequencing.

3.4 4C screening sites are potential binding sites of CTCF

In this paper, we selected the interaction with the promoter region of LCR and β-globin gene HBD site, and included the potential CTCF (CCCTC-binding factor) binding site as 4C target site.CTCF is a zinc finger protein with highly conserved structure and diverse functions, which is involved in the process of chromatin tissue and remodeling throughout the genome.According to the results of 4C sequencing analysis, the potential binding sites of CTCF were selected for further verification and functional analysis.The screening results are shown in Table 1, the down-regulated site AC105129.4 which interacts with LCR on chromosome 15 and the up-regulated site AL354707.17 on chromosome 9.The interaction with HBD is located at the down-regulation site AC078785.22 after chromosome 3 induction and at the up-regulation site AC021646.35 after chromosome 12 induction.

The results of quantitative PCR analysis by anti-CTCF chromatin immunoprecipitation (ChIP) are shown in figure 4.The enrichment efficiency of anti-CTCF ChIP to the four sites was different.Before induction, the enrichment efficiency of AC105129.4 and AC078785.22 sites was higher, while that of AL354707.17 and AC021646.35 sites was lower.After induction treatment, the enrichment efficiency of AC105129.4 and AC078785.22 decreased,while that of AL354707.17 and AC021646.35 increased.The difference was statistically significant (P＜0.05).It is suggested that AC105129.4, AL354707.17, AC078785.22 and AC021646.35 are potential binding sites for CTCF.

Tab 1 Selection of sequencing sites

Fig 4 Analysis of CTCF enrichment of selected sites before and after induction

3.5 Functional analysis of potential remote interaction sites of β-globin gene

According to the results of 4C-seq, the potential sites interacting with the promoter regions of LCR and HBD were selected as AC105129.4 on chromosome 15, AL354707.17 on chromosome 9,AC078785.22 on chromosome 3 and AC021646.35 on chromosome 12 (as shown in Table 1).

The interaction frequency between AC105129.4 and AC078785.22 was further verified by 3C technique.The results showed that there was a higher interaction frequency between AC105129.4 and AC078785.22 and the target site before induction (Uninduced), but decreased after induction (ATRA-induced) (figure 5A/5C).FAIRE analysis showed that the open region of the site was more open before induction, but decreased after induction (ATRA-induced)(figure 5B/5D).Previous studies in this paper showed that the expression of LCR and HBD genes was down-regulated and the opening degree of the site decreased in the process of differentiation induced by early progeny.The verification results of AC105129.4 and AC078785.22 loci were consistent with the results of sequencing and gene expression.The results of AL354707.17 and AC021646.35 loci were opposite to those of gene expression.The differences were statistically significant (P＜0.05).

The Epiregio online web tool can be used to verify the existence of regulatory elements in genes or genomic regions of interest [16].In this paper, the functional analysis of the regulatory elements in the genomic region of the locus was carried out through the Epiregio web page.As shown in Table 2, the regression coefficient of regulatory elements contained in AC105129.4 and AC078785.22 is greater than zero, indicating functional activation of regulatory elements (activating).The regression coefficient of AL354707.17 and AC021646.35 is less than zero, indicating the functional inhibition of regulatory elements (repressing).The predicted results are basically consistent with the previous verification results, indicating that AC105129.4, AL354707.17, AC078785.22 and AC021646.35 are important potential regulatory elements and have important effects on gene expression.

Fig 5 Analysis of interaction sites with LCR and HBD promoter region

Tab 2 Prediction of regulatory element Information interacting with LCR and HBD promoter region

4.Discussion

The expression and transformation of β-globin gene is a complex regulatory network involving a variety of regulatory elements.At present, studies have shown that the upstream LCR is involved in the regulation of β-globin gene family expression [25,26].In addition to LCR, there are other remote regulatory elements involved in the regulation of β-globin gene expression.4C method is combined with high-throughput sequencing to screen the sites that interact with anchor sites in the whole genome.In the process of 4C nested reverse PCR amplification, in order to increase the sensitivity and diversity of 4C PCR amplification, it is necessary to optimize the PCR amplification conditions[27,28].The loci captured by 4C analysis are located on different chromosomes, and the sequencing frequency of these loci indirectly reflects the interaction frequency between each site and 4C target site to some extent[29,30].

In this paper, the dynamic expression and regulation mechanism of β-globin family genes before and after induction of promyelocyte differentiation was analyzed.The promoter region and LCR of each gene in the β-globin locus were used as 4C anchor sites to screen the sites that interact with 4C anchor sites in the whole genome.The results show that the region of interaction with β-globin gene occurs not only within the chromosome, but also other unknown regulatory elements participate in the long-distance interaction between chromosomes.The sequencing results show that AC105129.4 and AC078785.22 bind to the target site efficiently before induction, indicating that these two sites may have important regulatory elements to enhance site interaction.The interaction frequency and enrichment of AL354707.17 and AC021646.35 were up-regulated after induction, indicating that there may be important regulatory elements for the interaction of inhibitory sites.Through ChIP analysis, the results showed that the four sites could be effectively enriched by anti-CTCFChIP before and after induction treatment.Therefore, 4C screening sites AC105129.4, AL354707.17,AC078785.22 and AC021646.35 are potential binding sites for CTCF.3C and FAIRE techniques were used to further verify that there may be important regulatory elements interacting with LCR and HBD at the four sites, and then the potential functions of regulatory elements were analyzed.Through the functional analysis of the regulatory elements in the genomic region where the locus is located, it is proved that AC105129.4, AL354707.17, AC078785.22 and AC021646.35 are important potential regulatory elements.And after induced differentiation, the interaction frequency and enrichment between AL354707.17 and AC021646.35 regulatory elements were up-regulated, contrary to gene expression, there may be inhibition, showing the function of Silencer.Through the functional verification of regulatory elements, it is of great significance to study the β-globin family gene loci in the genomewide interaction network, and lay a foundation for further exploring the dynamic expression and regulation mechanism of β-globin family genes.

Important statement

All the authors read and understand the policy statement in the Journal of Hainan Medical College, and there is no potential conflict of interest in this study.

Author's contribution

Xiao Yi-shu is responsible for part of the research and article writing; Xu Lan and Liu Chun-ya are responsible for part of the research work; Du Le, Sun Yuan-tian and Li Dong-na participate in part of the experimental guidance; Ren Li-cheng is responsible for this research organization, technical guidance, and guiding the writing of this article.

All the authors declare no conflict of interests.