The Chicken HDAC4 Promoter and Its Regulation by MYC and HIF1A

Abstract

Background: Histone deacetylase 4 (HDAC4) is a member of the class II histone deacetylase family, whose members play a crucial role in various biological processes. An in-depth investigation of the transcriptional characteristics of chicken HDAC4 can provide fundamental insights into its function.

Methods: We examined HDAC4 expression in chicken embryonic stem cells (ESC) and spermatogonial stem cells (SSC) and cloned a 444 bp fragment from upstream of the chicken HDAC4 transcription start site. Subsequently, we constructed pEGFP-HDAC4 and a series of 5'-deletion luciferase reporter constructs, which we transfected into DF-1 cells to measure their transcriptional activity. The regulatory mechanisms of chicken HDAC4 expression were investigated by performing trichostatin A (TSA) treatment, deleting putative transcription factor binding sites, and altering transcription factor expression levels.

Results: HDAC4 exhibited higher expression in SSC than in ESC. We confirmed that the upstream region from -295 bp to 0 bp is the core transcriptional region of HDAC4. TSA effectively inhibited HDAC4 transcription, and bioinformatics analysis indicated that the chicken core HDAC4 promoter sequence exhibits high homology with those of other avian species. The myelocytomatosis viral oncogene homolog (MYC) and hypoxia-inducible factor 1 α (HIF1A) transcription factors were predicted to bind to this core region. Treatment with TSA for 24 h resulted in the upregulation of MYC and HIF1A, which repressed HDAC4 transcription.

Conclusions: Our results provide a basis for subsequent investigations into the regulation of HDAC4 expression and biological function.

Keywords: HDAC4; HIF1A; chicken; transcriptional regulation.

Figure 1

HDAC4 promoter core region analysis. (A): Online prediction of the HDAC4 promoter region. Different color boxes correspond to the promoter positions predicted by different prediction software. TA means the predicted TATA box. E means the predicted enhancer. (B): Full-length amplification of the HDAC4 promoter (including PEGFP-N1 homologous arms). (C): Schematic diagram and sequencing chromatogram of the pEGFP-HDAC4 vector construction. (D): Validation of pEGFP-HDAC4 activity, DF-1 cells expressing green fluorescence. (E): The activity of each vector was measured by a dual-luciferase assay. Note: PGL3-P1: Fragment 1 (−166 bp to +1 bp, chr7:6467299-6467132) linked to the PGL3-Basic vector, PGL3-P2: Fragment 2 (−295 bp to +1 bp, chr7:6467428-6467132) linked to the PGL3-Basic vector, PGL3-P3: Fragment 3 (−444 bp to +1 bp, chr7:6467577-6467132) linked to the PGL3-Basic vector. ns: no significant difference, **: p < 0.01.

Figure 2

TSA inhibited HDAC4 transcription. (A): qRT-PCR analysis of the expression of HDAC4 in DF-1 after TSA treatment with different concentrations for 24 h and 48 h. (B): The activity of each vector was measured by a dual-luciferase assay after TSA treatment with different concentrations for 24 h. *: p < 0.05, **: p < 0.01.

Figure 3

Bioinformatics analysis of the HDAC4 core promoter region. (A): Homology analysis of the upstream −295 bp to +1 bp region of HDAC4 transcription start site in different species. (B): Prediction of transcription factors putative binding to the upstream −295 bp to +1 bp region of chicken HDAC4 transcription start site. (C,D): qRT-PCR analysis of the expression of HIF1A and MYC in DF-1 after TSA treatment with different concentrations for 24 h and 48 h. *: p < 0.05, **: p < 0.01.

Figure 4

Effect of HIF1A and MYC putative binding sites on HDAC4 promoter activity. (A): Sequence of transcription factor putative binding sites of HIF1A and MYC. (B): Schematic diagram of deletion vectors for the putative binding sites of HIF1A and MYC. (C): The activity of each MYC/HIF1A putative binding site deletion vector was measured by a dual-luciferase assay. ns: no significant difference, **: p < 0.01.

Figure 5

HIF1A and MYC inhibit HDAC4 transcription. (A,B): qRT-PCR analysis of the expression of MYC and HDAC4 in DF-1 after transfection with KD-MYC (MYC knockdown vector) or OE-MYC (MYC overexpression vector). (C,D): qRT-PCR analysis of the expression of HIF1A and HDAC4 in DF-1 after transfection with KD-HIF1A (HIF1A knockdown vector) or OE-HIF1A (HIF1A overexpression vector). (E): The activity of PGL3-P2 was measured by dual-luciferase assay after transfection with KD-MYC or KD-HIF1A. (F): The luciferase activity of PGL3-P2 was measured by a dual-luciferase assay after transfection with OE-MYC or OE-HIF1A. *: p < 0.05, **: p < 0.01.