An NAD+-dependent novel transcription factor controls stage conversion in Entamoeba
- Stanford University School of Medicine, United States
Figures
Figure 1 with 2 supplements
Encystation regulatory motif (ERM) specifically binds cyst nuclear protein and identification of crucial residues important for promoter activity.
(A) Sequence logo of ERM, which is enriched in the promoter of cyst-specific genes. The seven-nucleotide motif information content is shown. (B) Representative EMSA results are shown in the presence …
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Figure 1—source data 1
List of genes specifically upregulated during 24 hr of encystation.
616 genes were identified which coordinately upregulated at 24 hr of Encystation. Gene ID and normalized expression from different time points of development are listed (Excel file).
- https://doi.org/10.7554/eLife.37912.005
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Figure 1—source data 2
List of oligonucleotides used in electrophoretic mobility shift assay (EMSA), cloning and RT-PCR.
All the primers used in this study for EMSA, cloning and RT-PCR are listed with gene ID (Excel file).
- https://doi.org/10.7554/eLife.37912.006
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Figure 1—source data 3
List of genes having ERM (CAACAAA) in the promoter.
A total of 131 genes were identified which have CAACAAA motif in the promoter regions (Excel file).
- https://doi.org/10.7554/eLife.37912.007
Figure 1—figure supplement 1
Motifs enriched in the promoters of genes that upregulated during 24 hr of encystation.
Nine motifs (Motif 1 – 9) were identified which enriched in the promoters of cyst-specific genes compare to all promoter databases in E. invadens. Motif name, number of occurrences within …
Figure 1—figure supplement 2
Summary of screening results of the motifs by electrophoretic mobility shift assay (EMSA).
EMSA by using radiolabeled oligonucleotides for all nine motifs with nuclear extract from both trophozoites and cysts (24 hr). Each unlabeled oligonucleotide at 10X and 100X was used as a specific …
Figure 2 with 2 supplements
Confirmation of ERM-binding protein (ERM-BP).
(A) EMSA results with purified GST tagged ERM-BP and radiolabeled ERM probe. Unlabeled ERM oligonucleotides in 100X excess were used as a specific competitor and GST as control protein. The red …
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Figure 2—source data 1
All proteins identified from three independent mass-spec experiments.
Mass spectrometry was performed on three paired samples (ERM-WT and ERM-core). In one experiment, ERM-WT with trophozoite nuclear extract was used as a second control. The cut-off criteria: minimum protein, 95%; minimum number of peptides, 1; minimum peptide, 95% were used for analysis. All the proteins identified are listed with Gene ID, spectral count and fold change (Excel file).
- https://doi.org/10.7554/eLife.37912.012
Figure 2—figure supplement 1
Summary of mass-spec results.
LC-MS data analysis from three independent experiments is shown. In parenthesis the numbers proteins (168, 93 and 227), which were enriched or exclusively present in ERM-WT compare to ERM-core. Only …
Figure 2—figure supplement 2
Stage-specific expression of ERM-BP and confirmation by gel super-shift assay.
(A) RT-PCR to detect the expression of ERM-BP (EIN_083100) transcript level in E. invadens (IP1) trophozoites (Troph) and cysts (24 hr) with a loading control (EIN_327460) and an encystation control …
Figure 3
Myc-tagged ERM-BP is localized to nucleus and overexpression enhances encystation.
Immunostaining with α-myc antibody in (A) trophozoites, and (B) 24 hr cyst was performed in ERM-BP_OX and control cells (Green). DNA was stained with DAPI (Blue). Scale bar for trophozoites and …
Figure 4 with 2 supplements
Silencing ERM-BP decreases encystation and leads to formation of ghost like cysts.
(A) Data represents the number of cysts in control and silenced-ERM-BP cell lines after 72 hr of encystation. The number of cysts in control parasites was compared to silenced ERM-BP by calcofluor …
Figure 4—figure supplement 1
ERM-BP silencing using an RNAi-Trigger approach.
RT-PCR was performed to detect ERM-BP (EIN_083100) transcript level in control and silenced-ERM-BP cell lines (maintained at G418, 80 μg ml-1) from both trophozoites and 24 hr cyst with a loading …
Figure 4—figure supplement 2
ERM-BP regulates the expression of target genes.
(A) RT-PCR was performed to detect the expression of cyst-wall-specific genes (e.g. Jacob, Chitinase and Jessie) in control, silenced-ERM-BP and ERM-BP_OX cell lines. (B) RT-PCR in control …
Figure 5 with 2 supplements
Intracellular NAD+/NADH is elevated during encystation and NAD+ facilitates encystation.
(A) Measurement of intracellular NAD+/NADH in trophozoites (Troph) and 72 hr of cysts. Data are mean ±s.d. (n = 3) Student’s t-test; *p<0.05. (B) Intracellular NAD+ was detected from Entamoeba cells …
Figure 5—figure supplement 1
Intracellular NAD+ and NADH in trophozoites and sarkosyl resistant cysts.
The graph represents the actual concentrations of NAD+ and NADH in trophozoites (Trophs) and sarkosyl-resistant cysts in nmoles, per 2 × 106 cells. Data are mean ±s.d. (n = 3) Student’s t-test; *p<0.…
Figure 5—figure supplement 2
NAD+ but not NADH facilitates DNA binding.
Representative EMSA results with recombinant ERM-BP and radiolabeled ERM in presence of different concentrations of NAD+ or NADH (0, 1, 2, 4, 6, 8 and 10 mM). Gel shifted bands are shown by red arrow.
Figure 6 with 3 supplements
Protein sequence alignment and residues in ERM-BP important for NAM binding and catalytic activity.
Protein sequence alignment of ERM-BP from two Entamoeba species (E. invadens- EIN_083100 and E. histolytica- EHI_146360) and bacterial nicotinamidase (pncA) from Mycobacterium tuberculosis (Mt) and A…
Figure 6—figure supplement 1
EMSA with recombinant proteins in the presence of varying concentrations of NAD+.
Representative EMSA results by using recombinant ERM-BP-WT and mutant proteins (ERM-BP-DBM, C198A, K150A and D12A) with radiolabeled ERM in presence of different concentrations of NAD+ (0, 1, 2, 3 …
Figure 6—figure supplement 2
Thermal stability of ERM-BP-WT and mutants in presence of different concentrations of NAD+.
The fluorescence intensity (FI) at varying temperatures (upper panel) and derivative melt curves calculated by differences in FI at each temperature (lower panel) are shown. Peak temperature in the …
Figure 6—figure supplement 3
Enzymatic activity of ERM-BP-WT and mutants.
The graph represents percentage of nicotinamide turn over into nicotinic acid by ERM-BP-WT and mutant recombinant proteins. Bacterial PncA was used as positive control and GST as a negative control. …
Figure 7
Proposed model for the role of ERM-BP in encystation.
The transcription factor ERM-BP binds to NAD+ and undergoes a conformational change, which facilitates the binding to the CAACAAA motif in the promoter of cyst-specific genes. ERM-BP also catalyzes …
Tables
Table 1
Summary of proteins identified by LC-MS and confirmation.
Six proteins were identified in all three experiments as enriched in the ERM-WT sample and shown here with Gene ID and annotations. All six proteins were expressed in bacteria and the only …
| No | Gene ID | Proteins | Recombinant protein bind to ERM | Overexpression in Amoeba | Localization |
|---|---|---|---|---|---|
| 1 | EIN_212680 | WD-repeat protein | No | Yes | Cytoplasm |
| 2 | EIN_192460 | hypothetical | No | ND | |
| 3 | EIN_224750 | Protein-tyrosine phosphatase | No | ND | |
| 4 | EIN_224120 | Hypothetical | No | Yes | Cytoplasm |
| *5 | EIN_083100 | Hypothetical | Yes | Yes | Nucleus/ Cytoplasm |
| *6 | EIN_024000 | Hypothetical | No | ND |
Table 2
DNA binding, NAD+ binding and catalytic activity of ERM-BP-WT and ERM-BP mutants.
Wild type (WT) and mutant ERM-BP proteins were analyzed by three approaches. DNA binding was determined by EMSA analysis with increasing concentration of NAD+. Protein thermal shift assay was …
| Proteins | DNA Binding (EMSA) NAD+ (mM)# | NAD+ Binding (Tm) NAD+ (mM)§ | Nicotinamidase activity (% conversion)¶ | |||||
|---|---|---|---|---|---|---|---|---|
| 0 | 1 | 4 | 0 | 1 | 4 | P-values (0-4) | ||
| 1. GST† | - | - | - | 52 ± 7.0 | 53 ± 7.0 | 51 ± 7.0 | 0.6549 | no conversion |
| 2. ERM-BP-WT‡ | ++ | +++ | ++++ | 57 ± 0.3 | 60 ± 0.7 | 62 ± 1.3 | 1.1E-08 | 52 ± 0.6 |
| 3. D12A‡ | +/- | ++ | +++ | 57 ± 1.0 | 59 ± 1.0 | 62 ± 1.0 | 1.5E-08 | no conversion |
| 4. ERM-BP-DBM‡ | - | - | ++ | 57 ± 0.3 | 58 ± 0.8 | 61 ± 1.0 | 9.9E-09 | 50 ± 2.1 |
| 5. K150A‡ | +/- | ++ | +++ | 58 ± 1.7 | 59 ± 1.0 | 62 ± 11 | 3.7E-05 | no conversion |
| 6. C198A† | - | - | -/+ | 57 ± 0.3 | 57 ± 0.4 | 57 ± 0.5 | 0.3796 | no conversion |
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# EMSA (Electrophoretic mobility shift assay) with varying amounts of NAD+ (0, 1, 4 mM): ‘++++' indicates strong binding, ‘+++' and ‘++' indicate moderate, ‘+/-' indicates weak binding and '-'“indicates no binding. § NAD+ binding was monitored by protein thermal shift assay with varying amounts of NAD+ (0, 1, 4 mM). The Tm (melting temperature) is shown as mean ±s.d. (n = 3) Student’s t-test; p-values are shown between NAD+ 0 mM and 4 mM concentrations. ¶ The turnover of nicotinamide to nicotinic acid by ERM-BP-WT and mutant recombinant proteins are shown as percentage conversion mean ±s.d. (n = 3). ‡ Indicates having significant effects in DNA /NAD+ binding and catalytic activity, † Indicates not having significant effects in DNA/NAD+ binding and catalytic activity.
Table 3
Summary of DNA binding, NAD binding and enzymatic activity of wild type and mutant versions of ERM-BP are shown.
DNA binding, NAD+ binding and enzymatic activity of ERM-BP-WT and ERM-BP mutants are summarized. ‘+' indicates binding or having enzymatic activity and '-' indicates no binding or no activity.
| Proteins | DNA Binding | NAD+ binding | Enzymatic activity |
|---|---|---|---|
| ERM-BP-WT | + | + | + |
| D12A | + | + | - |
| ERM-BP-DBM | - | + | + |
| K150A | + | + | - |
| C198A | - | - | - |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.37912.027
