Characterization

Description(s):

To characterize transcription factors (TFs) that binds to a specific promoter or that regulate the expression of a specific gene via its upstream promoter, two common approaches are applied. First is to employ gel shift assay with DNA binding sites of TFs that are silico-identified within the promoter. Second is to removal or knockout the binding site(s) of a specific TF in order to measure whether the expression of a promoter-linked reporter is increased or decreased. Often, a series of reporter constructs with the promoter deletions or mutations need to make because many binding sites of one or a few TFs are present within a promoter. Signosis has developed a fast method to facilitate the characterization of promoters through a revised TF activation array. This assay will help to test whether a selected 96 TFs bind to the promoter or not.

This kit also includes our Nuclear Extraction Kit. This kit is used for preparation of nuclear extracts, which is often needed for studying transcription factors. The Nuclear Extraction Kit contains reagents for the preparation of 200 nuclear extracts from culture cells in 6-well plate and 50 nuclear extracts from cultured cells in 100-mm culture dishes. The kit can also be used for nuclear extraction of soft tissue samples (brain, kidney, liver, etc); however, there may be additional steps required to release cells from tissues.

Applicable Grid:

List of Applicable TFs:

1

2

3

4

5

6

7

8

9

10

11

12

A

AP1

CDP

GATA

NF-1

Pit

Stat3

XBP

FOXG1

HoxA-5

NRF2(A)

ProX1

SOX2

B

AP2

CREB

GR/PR

NFAT

PPAR

Stat4

AP3

FOXO1

HSF

Oct-1

RB

SOX9

C

AR

E2F-1

HIF

NF-E2

PXR

Stat5

AP4

FREAC-2

KLF4

Pax2

RUNX

SOX18

D

ATF2

EGR

HNF4

NFkB

SMAD

Stat6

COUP-TF

Gfi-1

MyoD

Pax3

ROR(RZR)

TFE3

E

Brn-3

ER

IRF

OCT4

Sp1

TCF/LEF

ELK

Gli-1

MZF

Pax8

RXR

TFEB

F

C\EBP

Ets

MEF2

p53

SRF

TR

FOXA1

HEN (NSCL-1)

Nkx2-5

PIT1

SF-1

USF-1

G

CAR

FAST-1

Myb

Pax-5

SATB1

YY1

FOXC1

HNF-1

Nkx3-2

PLAG1

SMUC

VDR

H

CBF

GAS/ISRE

Myc-Max

Pbx1

Stat1

TFIID

FOXD3

HOX4C

NRF1

MEF1

Snail

WT1

 

Principle

Promoter-binding TF profiling plate arrays are a competition assay of Signosis’ TF activation plate arrays. In the TF activation plate arrays, if all of 48 or 96 targeted transcription factors exist in the assayed samples, they will form 48 or 96 types of complexes, each TF with its corresponding biotin-labeled oligo (just like the complex in the gel shift assay). After a simple spin separation of the complexes from unbound free biotin-labeled oligos with a membrane-based column, TF-bound oligos eluted from the column and used for plate hybridization in which a complementary DNA of biotin-labeled oligo. The captured oligo is then detected with streptavidin-HRP and a chemiluminescent substrate. If any TF is not present, it will not form a complex, leading to no detection of TF in the following plate assay of biotin-labeled oligo. In promoter-binding TF profiling arrays, PCR fragment containing the promoter of your interest is mixed with a set of 48 or 96 biotin-labeled oligos corresponding to 48 or 96 TFs along with an assayed sample. If unlabeled promoter DNA fragment contains a TF binding sequence, it will compete with the biotin-labeled oligo to bind to the TF in the sample, leading to no or less biotin labeled TF/DNA complex formation and no or lower detection. Through comparison in the presence and absence of the competitor promoter DNA fragment, promoter-bound TFs can be identified.

Data

Promoter-Binding TF Profiling Assay: HeLa cells were treated with or without PMA. PMA was used to acticve TFs including AP1 and NFkB. Nuclear extracts were prepared and incubated with TF binding oligo probe mix: control HeLa cells without PMA treatment with the probe mix (blue); PMA-treated HeLa cells with the probe mix alone (red) and the probe mix plus IL6 promoter DNA fragment (yellow).

Literature

View user manual

Citations

1. Decoding Molecular Interplay between RUNX1 and FOXO3a Underlying the Pulsatile IGF1R Expression during Acquirement of Chemoresistance. Dhadve, Ajit C., Kishore Hari, Bharat Rekhi, Mohit Kumar Jolly, Abhijit De, and Pritha Ray.  Biochimica Et Biophysica Acta (BBA) - Molecular Basis of Disease (2020): 165754.
2. Cross-talk between macrophages and atrial myocytes in atrial fibrillation. Zewei SunDongchen ZhouXudong XieShuai WangZhen WangWenting ZhaoHongfei XuLiangrong Zheng. Basic Research in Cardiology, 2016. DOI: 10.1007/s00395-016-0584-z
3. Snail-activated long non-coding RNA PCA3 up-regulates PRKD3 expression by miR-1261 sponging, thereby promotes invasion and migration of prostate cancer cells. JH He, BX Li, ZP Han, MX Zou, L Wang, YB Lv. Tumor Biology, 2016 Original Article First Online: 14 October 2016. DOI: 10.1007/s13277-016-5450-y
4.  Myocyte enhancer factor (MEF)-2 plays essential roles in T-cell transformation associated with HTLV-1 infection by stabilizing complex between Tax and CREB. P Jain, A Lavorgna, M Sehgal, L Gao, R Ginwala.  Retrovirology, 2015Jain et al. Retrovirology(2015) 12:23 DOI 10.1186/s12977-015-0140-1
5. Functional and Activity Analysis of Cattle UCP3 Promoter with MRFs-Related Factors. Chen, W., Xu, H., Chen, X., Liu, Z., Zhang, W., & Xia, D. (2016). IJMS International Journal of Molecular Sciences, 17(5), 682. doi:10.3390/ijms17050682
6. Up-Regulation of CREG Expression by the Transcription Factor GATA1 Inhibits High Glucose- and High Palmitate-Induced Apoptosis in Human Umbilical Vein Endothelial Cells. Liu, Y., Tian, X., Li, Y., Liu, D., Liu, M., Zhang, X., Han, Y. (2016). PLOS ONE PLoS ONE,11(5). doi:10.1371/journal.pone.0154861
7. Characterization of the NLRC5 promoter in chicken: SNPs, regulatory elements and CpG islands. XM Guo, XP Liu, GB Chang, L Xu, YL Bi, HZ Wang DOI: 10.1111/age.12450 Animal Genetics, 2016
8. C3 exoenzyme impairs cell proliferation and apoptosis by altering the activity of transcription factors
   L von Elsner, S Hagemann, I Just, A Rohrbeck DOI: 10.1007/s00210-016-1270-2 Naunyn-Schmiedeberg's Archives of Pharmacology 2016
9. MGMT DNA repair gene promoter/enhancer haplotypes alter transcription factor binding and gene expression
   M Xu, CE Cross, JT Speidel, SZ Abdel-Rahman doi:10.​1007/​s13402-016-0286-4 Cellular Oncology, 2016
10. Overexpression of Long Non-coding RNA MEG3 Inhibits Proliferation of Hepatocellular Carcinoma Huh7 Cells via Negative Modulation of miRNA-664. Jin-Hua He, Ze-Ping Han, Ji-Ming Liu, Jia-Bin Zhou, Mao-Xian Zou, Yu Bing Lv, Yu-Guang Li, Ming-Rong Cao. Journal of Cellular Biochemistry. 2017 April 4. doi: 10.1002/jcb.26018.