Identification of a novel distal regulatory element of the human Neuroglobin gene by the chromosome conformation capture approach

Abstract

Neuroglobin (NGB) is predominantly expressed in the brain and retina. Studies suggest that NGB exerts protective effects to neuronal cells and is implicated in reducing the severity of stroke and Alzheimer's disease. However, little is known about the mechanisms which regulate the cell type-specific expression of the gene. In this study, we hypothesized that distal regulatory elements (DREs) are involved in optimal expression of the NGB gene. By chromosome conformation capture we identified two novel DREs located -70 kb upstream and +100 kb downstream from the NGB gene. ENCODE database showed the presence of DNaseI hypersensitive and transcription factors binding sites in these regions. Further analyses using luciferase reporters and chromatin immunoprecipitation suggested that the -70 kb region upstream of the NGB gene contained a neuronal-specific enhancer and GATA transcription factor binding sites. Knockdown of GATA-2 caused NGB expression to drop dramatically, indicating GATA-2 as an essential transcription factor for the activation of NGB expression. The crucial role of the DRE in NGB expression activation was further confirmed by the drop in NGB level after CRISPR-mediated deletion of the DRE. Taken together, we show that the NGB gene is regulated by a cell type-specific loop formed between its promoter and the novel DRE.

Figure 1.

(A) NGB mRNA level in three human cell lines quantified by qRT-PCR. The NGB level in SH-SY5Y cells was 300-fold of that in HeLa cells and 240-fold of that in K562 cells. ***P < 0.001 compared to HeLa (N = 3, mean ± SEM). (B and C) The 3C profiles in SH-SY5Y and HeLa cells of the 300 kb region surrounding the NGB gene. The anchor fragments are indicated by the dashed lines. Approximate positions and direction of transcription of genes, and EcoRI cutting sites within the area are displayed at the top. Significant differences between cell types were detected by Student's t test (*P < 0.05) (N = 3, mean ± SEM).

Figure 2.

(A) Modified screen shot of UCSC genome browser page showing DHS and ChIP-seq data of the two EcoRI fragments interacting with the NGB anchor fragment. The upstream fragment (chr14:77 806 771–77 809 773 (hg19)) contains Element I used for luciferase assays. (B) The downstream fragment (chr14:77 635 927–77 639 784 (hg19)) contains Element II. The elements used for luciferase assays are outlined by rectangular boxes. (C and D) Relative luciferase activities of the constructs in SH-SY5Y and HeLa cells. On the left, structures of the constructs used for luciferase assays are shown. The white boxes represent Element I (C) and Element II (D) respectively. The arrows in the box indicate the orientation of the inserted element. All the data are normalized to the promoter-only construct. ***P < 0.001 compared to promoter-only construct, ^#P < 0.05 between groups (N = 6, mean ± SEM).

Figure 3.

GATA factors binding revealed by ChIP. The enrichments of the target proteins at specified sites are expressed relative to that of non-specific immunoprecipitated (IP) DNA by rabbit IgG. The binding of GATA-2 (A) but not GATA-3 (B) was observed in SH-SY5Y cells. *P < 0.05, **P < 0.01 for comparing enrichment to the signal obtained with rabbit IgG IP (N = 5, mean ± SEM).

Figure 4.

(A) The mRNA level of GATA-2, GATA-3 and NGB upon GATA factor knockdown. cDNA was synthesized from total RNA extracted from untreated SH-SY5Y cells (Control), and cells infected with control shRNA vector (SHC002) or GATA-2 (pLKO-G2a and pLKO-G2b) or GATA-3 (pLKO-G3a and pLKO-G3b) targeting shRNA constructs. The amount of mRNA is normalized to β-actin level, and expressed relative to the level in control SH-SY5Y cells. *P < 0.05, **P < 0.01, ***P < 0.001 compared to SHC002 SH-SY5Y cells (N = 3, mean ± SEM). (B) Western blot analyses of GATA-2 and -3 knockdown SH-SY5Y cells. Proteins were extracted from untreated SH-SY5Y cells (Control), and cells infected with control shRNA vector (SHC002) or GATA-2 (pLKO-G2a and pLKO-G2b) or GATA-3 (pLKO-G3a and pLKO-G3b) targeting shRNA constructs. Bands shown are from three different membranes (one for control and SHC002, one for pLKO-G2a and b, and one for pLKO-G3a and b). The amount of protein loaded and exposure times were kept constant for each membrane. (C) mRNA level of NGB in SH-SY5Y cells with and without Element I deletion. Del-A and -B are cells with Element I deleted by different gRNA pairs. The levels are normalized to β-actin level and expressed relative to the level in control SH-SY5Y cells. *P < 0.05, **P < 0.01 compared to control SH-SY5Y cells (N = 3, mean ± SEM).

Figure 5.

Microarray analysis of GATA-2 and NGB expression level in different parts of the brain (Image credit: Allen Institute (53)). Associated expression of the two genes is shown in dorsal and ventral thalamus, mesencephalon, pontine tegmentum and myelencephalon. Red and green on the heat map represent high and low expression respectively. For each structure, six samples are displayed.

Figure 6.

Schematic diagrams illustrating the function of Element I in NGB gene transcription regulation. (A) In NGB-expressing cells, a chromatin loop is formed between the NGB promoter and Element I. The loop brings GATA-2 on Element I and Sp1 on the hypomethylated NGB promoter (open circle) in close proximity and activates transcription. (B) In cells not expressing NGB, the promoter of NGB gene is hypermethylated (filled circles) and binding of Sp1 and Sp3 is inhibited. This impairs the interaction between the promoter and Element I. The formation of looping may also be affected by competition between GATA-2 and -3 for the binding sites within Element I.