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Investigation of Possible Second Promoter in Cd4 through Comparison of the Relative Abundance of Transcripts

Colby Uptegraft and Sophia SarafovaBiology Department, Davidson College, Davidson, NC 28036

1Siu, G (2002). Immunology. 14: 441-451.

Cd4 Locus

Background

The lineage commitment of T cells as they develop from double-positive (DP) (CD4+CD8+) to CD4+ single-positive (SP) thymocytes requires the maintenance of Cd4 gene expression. The mechanisms controlling CD4 expression have been determined to be primarily transcriptional, and the locus in mice (Mus musculus) has been characterized to include a silencer, promoter (P1), thymocyte enhancer (TE), distal enhancer (DE), and a proximal enhancer (PE).1 The human Cd4 gene locus contains an additional promoter (P2), and this corresponding region in mice has yet to be fully investigated. A second promoter in mice would help explain the sustained CD4 signal throughout T cell maturation and the different surface CD4 protein levels between DP and SP cells. If P2 exists, then two transcripts will be present, and their relative steady-state amounts can be compared through quantitative RT-PCR in ex vivo derived and cultured cells.

DE PE P1 Ex1 S Ex2

ATG

Ex3 Ex4 Ex6Ex5 Ex9Ex8 Ex10Ex7 TEP2

???

Ex1 Ex2

ATG

Ex3 Ex4 Ex5 Ex6 Ex7 Ex8 Ex9 Ex10

Processed P1 mRNA

Processed P2 mRNA?

Ex2 Ex3 Ex4 Ex5 Ex6 Ex7 Ex8 Ex9 Ex2

ATG

Ex3 Ex4 Ex5 Ex6 Ex7 Ex8 Ex9 Ex10

RNA Isolation

RLM-11AKR1G1

DP Cell Lines

Thymus Cells

Lymph Node Cells

FACS Staining

CD8α antibodyTagged with FITC

CD4 antibodyTagged with PE

For Cell Lines and Thymus:

For Lymph Node:

Dynal Bead CD4 SP Isolation

PNA Panning

PNABeads Magnet

CD4 SP in Supernatant

DP bound to PNA

Post PurificationFACS Staining

CD4 antibodyTagged with PE

TCRβ antibodyTagged with FITC

TRIzol®

Reverse Transcription and qPCRLN CD4 SP

RNATHY DP RNA RLM-11 RNA AKR1G1 RNA

DNase Treatmentand

RNA Cleanup

ReverseTranscription

(cDNA Synthesis)

AAAAA AAAAA AAAAA AAAAA

TTTTT TTTTT TTTTT TTTTT

Oligo dT primers bind to Poly A tails of mRNA.

AAAAA

AAAAAGGG

GGG

RNase Treatment,PCR, and

qPCR

DNase treatment followed by Qiagen’s RNA cleanup kit removes contamination.

With the same reverse primer, two unique forward primers and DNA polymerase amplify regions unique to P1 and P2 cDNA. SYBR green, a strong fluorescent intercalating agent, detects dsDNA and allows for the relative quantization of initial transcript levels in qPCR.

P1

P2

RV

P1

P2

RV

Primer Design

SYBR Green

qPCR Detector

Ex1 Ex2

ATG

Ex3 Ex4 Ex5 Ex6 Ex7 Ex8 Ex9 Ex10

P1 cDNA P2 cDNA

Ex2 Ex3 Ex4 Ex5 Ex6 Ex7 Ex8 Ex9 Ex2

ATG

Ex3 Ex4 Ex5 Ex6 Ex7 Ex8 Ex9 Ex10

Exon 1 Exon 2 Exon 2

Intron 1 between P2 and Ex2

Reverse Primer

Reverse Primer

P1 Forward Primer

P2 Forward Primer

5’ CTG TGA AGG CAA AGC AAG ACT CTC 3’

Exon 3

5’ TCC CTT GAG TGA CAG CTA GGA GTT 3’

5’ TCT TAG TTT GGC AGG ACC TTT GGG 3’

Exon 3

5’ TCC CTT GAG TGA CAG CTA GGA GTT 3’

Expected P1-RV Amplicon Length: 231 bp

Expected P2-RV Amplicon Length: 303 bp

Figure 1A

B

1 2 3 4 5 6 7 8 9

1 2 3 4 5 6 7 8 9

1) 100 bp Ladder2) LN P1 & RV3) LN P2 & RV4) THY P1 & RV5) THY P2 & RV6) gDNA P2 & RV7) LN No RT8) THY No RT9) NTC

1) 100 bp Ladder2) RLM-11 P1 & RV3) RLM-11 P2 & RV4) RLM-11 GADPH5) RLM-11 No RT6) AKR1G1 P1 & RV7) AKR1G1 P2 & RV8) AKR1G1 GADPH9) AKR1G1 No RT

References

Future Directions

B

A

C

CTGTGAAGGCAAAGCAAGACTCTCTTCTTCACTAGGTACCTGTTTGCAAAGTCTCGAGCCCTCATATACACACACCTGCTGTGAAGGCAAAGCAAGACTCTCTTCTTCACTAGGTACCTGTTTGCAAAGTCTCGAGCCCTCATATACACACACCTGTGCAAGAAGCAGAGTGAAGGAAGGACTGGCCAGAGGCTCAGATTCCCAACCAACAAGAGCTCAAGGAGACCACCATGTGCAAGAAGCAGAGTGAAGGAAGGACTGGCCAGAGGCTCAGATTCCCAACCAACAAGAGCTCAAGGAGACCACCGTGTGCCGAGCCATCTCTCTTAGGCGCTTGCTGCTGCTGCTGCTGCAGCTGTCACAACTCCTAGCTGTCACTCAAGGGATGCCGAGCCATCTCTCTTAGGCGCTTGCTGCTGCTGCTGCTGCAGCTGTCACAACTCCTAGCTGTCACTCAAGGGA

RLM-11 P1 & RV

RLM-11 P2 & RV

Expected

Actual

TCTTAGTTTGGCAGGACCTTTGGGGTGCATTACTGCAGGGTGCCCACTTTTGTGTATGCAGATAATGTTTCTTAGTTTGGCAGGACCTTTGGGGTGCATTACTGCAGGGTGCCCACTTTTGTGTATGCAGATAATGTTCTCTGGGTTGGTTATCAAGGTCCTGAGGAAGAGAAAGAGTTCTTGTGTGCCCCAGGCCCAAGGAATACCTCTGGGTTGGTTATCAAGGTCCTGAGGAAGAGAAAGAGTTCTTGTGTGCCCCAGGCCCAAGGAATACCTGAAGACTGATGATTTCTATCTTCCTCCGCCCCTGACATTTTTGTAGGCTCAGATTCCCAACCAACAACTGAAGACTGATGATTTCTATCTTCCTCCGCCCCTGACATTTTTGTAGGCTCAGATTCCCAACCAACAAGAGCTCAAGGAGACCACCATGTGCCGAGCCATCTCTCTTAGGCGCTTGCTGCTGCTGCTGCTGCAGCTGAGCTCAAGGAGACCACCATGTGCCGAGCCATCTCTCTTAGGCGCTTGCTGCTGCTGCTGCTGCAGCTGTCACAACTCCTAGCTGTCACTCAAGGGAGTCACAACTCCTAGCTGTCACTCAAGGGA

• Devise more efficient method for purification of LN SP and THY DP cells.• Redesign P2 and RV primer pair to span larger intron gap or find better way to eliminate the gDNA contamination problem.

CD

8 (F

ITC

)

90.73%

5.07%TC

Rβ

(FIT

C)

CD

8 (F

ITC

)

CD4 (PE)

CD4 (PE)

93.93%

2.71%

CD4 (PE)

CD4 (PE)

TC

Rβ

(FIT

C)

40.68%

97.75%

CD

4 (P

E)

CD8 (FITC)

97.98%

CD8 (FITC)

CD

4 (P

E)

97.20%

Figure 2

Cd4 Transcript Levels Relative to GADPH

Two unique transcripts of the expected lengths were present in both B10.A LN SP and purified B6 THY DP cells. gDNA contamination in cDNA from both types of cells prevented qPCR analysis of the P2 transcript.

Two unique transcripts of the expected lengths were present in both DP thymoma cell lines as well, and GADPH primers yielded a product of the expected size. No gDNA contamination for either primer pair allowed for an accurate qPCR analysis.

Sequencing of the qPCR products from the RLM-11 cell line confirmed amplification of the correct targets.

DP thymoma cell lines showed similar amplification patterns for GADPH, P1, and P2 cDNA. GADPH cDNA is detected around cycle 20 while P1 and P2 cDNA are detected around cycles 25 and 30 respectively.

A significantly greater relative amount of the steady-state P1 as compared to the P2 transcript was present in both cell lines, suggesting P1 is the main promoter at the DP stage (P1:P2 ≈ 44 in RLM-11 and 67 in AKR1G1). The relative steady-state amount of P1 in B10.A LN SP cells was 2.25 times greater than the amount in purified DP THY cells. Which transcript is the predominant one remains to be determined.

AcknowledgementsWe would like to thank Chris Van Rooyen for the maintenance of our lab equipment, Amy Becton for the upkeep of our mice, Maria Cowen and Kay Filar for handling our many orders, Dr. Campbell’s lab for coping with our countless intrusions.