Regulatory effect of heat shock transcription factor-1 gene on heat shock proteins and its transcriptional regulation analysis in small abalone ... View Full Text


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Article Info

DATE

2020-11-24

AUTHORS

Xin Zhang, Yuting Li, Yulong Sun, Mingxing Guo, Jianjun Feng, Yilei Wang, Ziping Zhang

ABSTRACT

BackgroundThe effects of diverse stresses ultimately alter the structures and functions of proteins. As molecular chaperones, heat shock proteins (HSPs) are a group of highly conserved proteins that help in the refolding of misfolded proteins and the elimination of irreversibly damaged proteins. They are mediated by a family of transcription factors called heat shock factors (HSFs). The small abalone Haliotis diversicolor is a species naturally distributed along the southern coast of China. In this study, the expression of HdHSF1 was inhibited by RNAi in hemocytes in order to further elucidate the regulatory roles of HdHSF1 on heat shock responsive genes in abalone. Meanwhile, to understand the transcriptional regulation of the HdHSF1 gene, the 5′-upstream regulatory region of HdHSF1 was characterized, and the relative promoter activity was examined by dual-luciferase reporter gene assay system in HEK293T cell lines.ResultsAfter the inhibition of the H. diversicolor HSF1 gene (HdHSF1) by dsRNA (double-stranded RNA), the expression of most heat shock related-genes was down-regulated (p < 0.05). It indicated the importance of HdHSF1 in the heat shock response of H. diversicolor. Meanwhile, 5′-flanking region sequence (2633 bp) of the HdHSF1 gene was cloned; it contained a putative core promoter region, TATA box, CAAT box, CpG island, and many transcription elements. In HEK293T cells, the 5′-flanking region sequence can drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. Exposure of cells to the high-temperature (39 °C and 42 °C) resulted in the activation of HdHSF1 promoter activity, which may explain why the expression of the HdHSF1 gene participates in heat shock response. Luciferase activity of different recombinant plasmids, which contained different truncated promoter fragments of the HdHSF1 gene in HEK293T cells, revealed the possible active regions of the promoter. To further identify the binding site of the critical transcription factor in the region, an expression vector with the site-directed mutation was constructed. After being mutated on the GATA-1 binding site, we found that the luciferase activity was significantly increased, which suggested that the GATA-1 binding site has a certain weakening effect on the activity of the HdHSF1 promoter.ConclusionsThese findings suggest that GATA-1 may be one of the transcription factors of HdHSF1, and a possible signaling pathway mediated by HdHSF1 may exist in H. diversicolor to counteract the adverse effects of heat shock stress. More... »

PAGES

83

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/s12860-020-00323-9

DOI

http://dx.doi.org/10.1186/s12860-020-00323-9

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1132860885

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/33228519


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32 schema:description BackgroundThe effects of diverse stresses ultimately alter the structures and functions of proteins. As molecular chaperones, heat shock proteins (HSPs) are a group of highly conserved proteins that help in the refolding of misfolded proteins and the elimination of irreversibly damaged proteins. They are mediated by a family of transcription factors called heat shock factors (HSFs). The small abalone Haliotis diversicolor is a species naturally distributed along the southern coast of China. In this study, the expression of HdHSF1 was inhibited by RNAi in hemocytes in order to further elucidate the regulatory roles of HdHSF1 on heat shock responsive genes in abalone. Meanwhile, to understand the transcriptional regulation of the HdHSF1 gene, the 5′-upstream regulatory region of HdHSF1 was characterized, and the relative promoter activity was examined by dual-luciferase reporter gene assay system in HEK293T cell lines.ResultsAfter the inhibition of the H. diversicolor HSF1 gene (HdHSF1) by dsRNA (double-stranded RNA), the expression of most heat shock related-genes was down-regulated (p < 0.05). It indicated the importance of HdHSF1 in the heat shock response of H. diversicolor. Meanwhile, 5′-flanking region sequence (2633 bp) of the HdHSF1 gene was cloned; it contained a putative core promoter region, TATA box, CAAT box, CpG island, and many transcription elements. In HEK293T cells, the 5′-flanking region sequence can drive expression of the enhanced green fluorescent protein (EGFP), proving its promoter function. Exposure of cells to the high-temperature (39 °C and 42 °C) resulted in the activation of HdHSF1 promoter activity, which may explain why the expression of the HdHSF1 gene participates in heat shock response. Luciferase activity of different recombinant plasmids, which contained different truncated promoter fragments of the HdHSF1 gene in HEK293T cells, revealed the possible active regions of the promoter. To further identify the binding site of the critical transcription factor in the region, an expression vector with the site-directed mutation was constructed. After being mutated on the GATA-1 binding site, we found that the luciferase activity was significantly increased, which suggested that the GATA-1 binding site has a certain weakening effect on the activity of the HdHSF1 promoter.ConclusionsThese findings suggest that GATA-1 may be one of the transcription factors of HdHSF1, and a possible signaling pathway mediated by HdHSF1 may exist in H. diversicolor to counteract the adverse effects of heat shock stress.
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38 schema:keywords BackgroundThe effects
39 CAAT box
40 China
41 ConclusionsThese findings
42 CpG islands
43 GATA-1
44 H. diversicolor
45 HEK293T cell line
46 HEK293T cells
47 HSF1 gene
48 Haliotis diversicolor
49 RNAi
50 ResultsAfter
51 T cell lines
52 T cells
53 TATA box
54 abalone
55 abalone Haliotis diversicolor
56 activation
57 active region
58 activity
59 adverse effects
60 analysis
61 box
62 cell lines
63 cells
64 certain weakening effect
65 chaperones
66 coast
67 core promoter region
68 critical transcription factor
69 different recombinant plasmids
70 diverse stresses
71 diversicolor
72 dsRNA
73 dual-luciferase reporter gene
74 effect
75 elements
76 elimination
77 enhanced green fluorescent protein
78 exposure
79 exposure of cells
80 expression
81 expression vector
82 factor 1 gene
83 factors
84 family
85 findings
86 fluorescent protein
87 fragments
88 function
89 function of proteins
90 genes
91 green fluorescent protein
92 group
93 heat shock
94 heat shock factor
95 heat shock proteins
96 heat shock response
97 heat shock responsive genes
98 heat shock stress
99 hemocytes
100 importance
101 inhibition
102 islands
103 lines
104 luciferase activity
105 molecular chaperones
106 mutations
107 order
108 pathway
109 plasmid
110 promoter
111 promoter activity
112 promoter fragment
113 promoter function
114 promoter region
115 protein
116 putative core promoter region
117 recombinant plasmid
118 refolding
119 region
120 region sequences
121 regulation
122 regulation analysis
123 regulatory effects
124 regulatory regions
125 regulatory role
126 relative promoter activity
127 reporter gene
128 response
129 responsive genes
130 role
131 sequence
132 shock
133 shock factor
134 shock proteins
135 shock response
136 shock stress
137 site-directed mutations
138 sites
139 small abalone Haliotis diversicolor
140 southern coast
141 species
142 stress
143 structure
144 study
145 system
146 transcription elements
147 transcription factor 1 gene
148 transcription factors
149 transcriptional regulation
150 transcriptional regulation analysis
151 vector
152 weakening effect
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