Transcriptional regulation of drosophila Nanos

Transcriptional regulation of drosophila Nanos DR.IJAZ ALI Institute of Biotechnology and Genetic Engineering, Khyber Pakhtunkhwa Agricultural University, Peshawar

Outline • Introduction • Identification of the Nos minimal promoter and the effect of heterologous and nos 3’ UTRs on nosp in GSCs. • Identification of enhancer elements affecting Nosp in the germ line. • Nos and the DRE/DREF system of gene regulation. • A short promoter fragment confers expression on nos mRNA. • Conclusion.

Introduction • The Nanos gene • Conservancy of structure and function • Expression and role in germ line stem cells maintenance. • The nos promoter. • Confocal image of a germarium stained for Nos-Myc (red) and spectrosomes/fusomes (green). Nos is present in all GSCs, much reduced in dividing cysts, present at a high level in newly formed 16-cell cysts, and then down-regulated in older 16-cell cysts (Wang Z and Lin H. 2004).

Identification of the Nos minimal promoter and the effect of heterologous and nos 3’ UTRs on nosp in GSCsSoftware programs and websites • TF Search ver. 1.3. • Matinspector. • Mc006 TSS server (Duke university). • www.flyreg.org • www.ifti.org • www.epd.isb-sib.ch • http://www.ebi.ac.uk/emboss • Manual curetting

1. Continued…Computational analysis Table showing computational analysis of the promoter

1. continued…Computational analysis; CpG Islands CpG islands:regions of 200-1000 bp and a C+G content > 50%, occurs around transcription initiation sites, estimated frequency based on promoters in EPD: 39%. [Bucher, P. (2006). Comparative analysis of promoter sequences.] CpG Island/ EMBOSS/ Isochor result: Input sequence: 805 bp (From -680 to +125). The CpG Island: From -650 to -237 (413 bp).

1. Continued…Computational analysisThe transcription start site (TSS) MC006

Continued…Nosp 5’ deletion constructs for germ line transformation

1. Continued…Nosp 3’ deletion constructs for germ line transformation

1. Continued…Nosp:GFP constructs with various 3’ UTRs for germ line transformation • Nosp-680-GFP-nos 3’ UTR • Nosp-201-GFP-nos 3’ UTR • Nosp-80-GFP-bam 3’ UTR • Nosp-680-GFP-bam 3’ UTR • Nosp-78-GFP-nos 3’ UTR • Nosp-80-GFP-nos 3’ UTR

Continued…Nosp-GFP 5’ deletion lines by anti-GFP and Hts antibodies [Nosp-680/+125:GFP ] [Nosp-201/+103:GFP ]

1. Continued…Nosp-GFP 5’ deletion lines by anti-GFP and Hts antibodies [Nosp-80/+103:GFP ] [Nosp-78/+103:GFP ]

1. Continued…Nosp-GFP 5’ deletion lines by anti-GFP and Hts antibodies [Nosp-67/+103:GFP ] [Nosp-78/+103:GFP ] Line 2.

1. Continued…Nosp-GFP 5’ deletion lines by anti-GFP and Hts antibodies [Nosp-58/+103:GFP ] Line 1. [Nosp-58/+103:GFP ]Line 2.

1. Continued…Nosp-GFP 5’ deletion lines by anti-GFP and Hts antibodies Control [Nosp-47/+103:GFP ] Line 1.

1. Continued…Nosp-GFP 5’ deletion lines by anti-GFP and Hts antibodies [Nosp-32/+103:GFP ] Line 1. [Nosp-12/+103:GFP ] Line 1.

1. Continued…Nosp-GFP 5’ deletion lines by anti-GFP and Hts antibodies [Nosp-47/+103:GFP ] Line 2.

1. Continued…Nosp-GFP with various UTRs, Fly lines by anti-GFP and Hts antibodies [Nosp-78/+103:GFP-bam 3’ UTR ]. [Nosp-80/+103:GFP-nos 3’ UTR ]. [Nosp-78/+103:GFP-nos 3’ UTR ].

Continued…Summary of results Nosp 5’ deletions: List of constructs with respective expression in GSCs.

1. Continued…Summary of results Nosp 3’ deletions: List of constructs with respective expression in GSCs.

1. Continued…Summary of results Various 3’ UTRs driven by variable fragments of Nosp and their expression status:

2. Identification of enhancer elements affecting Nosp in the germ line

2. Continued…Internal deletions procedure for nosp Schematics show the procedure for internal deletions used for making [nosp-Del:GFP] constructs.

2. Continued…Nosp:GFP with internal deletions D4=-70 to -11 [Nosp-D4:GFP] Line 1 [Nosp-D4:GFP] Line 2

2. Continued…Nosp:GFP with internal deletions D2=-70 to +8 [Nosp-D2:GFP] Line 1 [Nosp-D2:GFP] Line 2

2. Continued…Nosp:GFP with internal deletions D6=-80 to +52 [Nosp-D6:GFP] Line 1 [Nosp-D6:GFP]Line 2

2. Continued… Summary of results Nosp internal deletion constructs and their respective GFP expression in the germ line stem cells.

2. Continued… The nos promoter from -680 to +125

3. Nanos and the DRE/DREF system of gene regulation DRE:DNA replication related element. DREF:DRE binding factor. DRE in nosp: -62 to -70. Eight bp palindromic (TATCGATA). Table shows primers for making constructs with deleted or mutated DRE

3. Continued…Nanos and the DRE/DREF system of gene regulation DRE mutation scheme (The overlap extention):

3. Continued…Nanos and the DRE/DREF system of gene regulation DRE= From -70 to -62 [Nosp-DRE:GFP] Line 1 [Nosp-DRE:GFP] Line 2

3. Continued…Nanos and the DRE/DREF system of gene regulation [Nosp-DRE:GFP] Line 2 [Nosp-DRE:GFP] Line 1 [Nosp-mut DRE:GFP] Line 2

3. Continued…Nanos and the DRE/DREF system of gene regulation DRE mutation= ATATGAAT [Nosp-mut DRE:GFP] Line 1 [Nosp-mut DRE:GFP] Line 3

3. Continued…DREF Cell clone analysis DREF (DNA replication related element factor):An 80 Kda homodimer Specific binding of DREF to DRE:An established fact (Hirose et al., 1993;Hirose et al., 2002; Ohno et al., 1993; Hocheimer et al., 2003; Hideki et al., 2004; Craig et al., 1999; Jamie et al., 2007). Clonal analysis • Recombination • ♂ neoFRT40A X ♀ Dkg 09294/cyo • ♀ Dkg.neoFRT40A X ♂ wi; sp/cyo • ♀/♂ neoFRT40A;?/cyo X ♂/♀ wi; sp/cyo • wi; Dkg.neoFRT40A/cyo (40 fly lines).

3. Continued…DREF Cell clone analysis b) FRT PCR to confirm recombination. Eight lines turned positive. • w hs-FLP; neoFRT40Aubi-gfp/cyo flies were crossed with DKG09294neoFRT40A/cyo to get the sample flies (w hs-FLP; neoFRT40AUbi-gfp/neoFRT40A.Dkg ). • The same genotype flies were crossed with neoFRT40A to get control flies (w hs-FLP; neoFRT40Aubigfp/neoFRT40A) for cell clone analysis. Result:

DREFRNAi • Dref (DNA replication related element factor) constructs and fly lines: • Dref Location: 2L, 30F2-30F3, 3.3 Kb. • Drefcontroledgenes:PCNA, ElF4e,DNA polalfa etc. 1) PUASP-dref-IR (Head to head) 2 lines 2) PUAS-Hs-dref 4 lines 3) PUASP-dref-IR {Tail to tail} 1 line 4) Hsp-dref-IR (head to head) 5) UAST-dref-IR (Head to Head) 6) UAST-dref-IR (Tail to Tail). Mechanism: Uas/Gal4 system. Methods: 1) Uast vector map) 2) Uasp vector map.

Novel RNAi construct making procedure

DREF RNAi • Heat shock and the mutant phenotype. • RNAi mutant X [Nosp-805-GFP] • IHC and GFP loss. • Electrophoretic mobility shift assay • Promoter fragment with DRE sequence • Labeling with radioactive isotope. • Nuclear extract • Incubation • Gel electrophoresis and documentation.

4. A short promoter fragment confers expression on Nos mRNA Rescue constructs: 1)Nosp-680-3XFlag-nos mRNA-nos 3’ UTR 2)Nosp-80- 3XFlag nos mRNA-nos 3’ UTR 3)Nosp-Del1- 3XFlag-nos mRNA-nos 3’ UTR 4)Nosp-Del2- 3XFlag-nos mRNA-nos 3’ UTR 5)Nosp-78-3XFlag –nos mRNA-nos 3’UTR 6)Nosp-201- 3XFlag-nos mRNA-nos 3’ UTR 7)Nosp-474- 3XFlag nos mRNA-nos 3’ UTR 8)Nosp-349-3X Flag-nos mRNA-nos 3’ UTR 9)Nosp-58-3XFlag-nos mRNA-nos3’ UTR E- box constructs

4. Continued…A short promoter fragment confers expression on Nos mRNA Schematics showing polyclonal site for making rescue constructs

4. Continued…A short promoter fragment confers expression on Nos mRNA Western blotting with Flag antibodies Nos-Flag fusion protein rescue by Western blotting. Shown in the figure are western blots for transgenes in the following order (1) Ote-3xflag-Ote (Positive control, 48.43 kda) (2) w1118 (negative control) (3) [Nosp-680- 3XFlag-nos mRNA-nos 3’UTR] (49.34 kda) (4) [Nosp-80-3XFlag-nosmRNA-nos3’UTR] (5) [Nosp-58-3XFlag-nosmRNA-nos 3’UTR] (6) [Nosp-Del2-3XFlag-nos mRNA-nos3’UTR].

4. Continued…A short promoter fragment confers expression on Nos mRNA Results for all 5’ and internal deletion constructs:

4. Continued…A short promoter fragment confers expression on Nos mRNA E-box and rescue:

6. Conclusion • The 5’margin of the nos minimal promoter is -58 relative to the transcription start site. • The 3’ limits of the minimal promoter is +52 relative to the transcription start site. • The nos minimal promoter sufficient for the transgenic GFP expression in the germ-line stem cells lay between position -58 and +52. • The 106 bp fragment has got all the regulatory elements sufficient for the transgene’s expression in the germ line stem cells. • The 11 bp sequence from -58 to -47 contain regulatory element essential for the transgene’s expression in the germ line stem cells. • Sequences downstream +52 are not required for the activity of Nosp in the germ-line and may be important for expression in cells other than the GSCs. • E-box does not regulate the transgene’s expression in GSCs. • The nos 3’ UTR does not execute any transcriptional regulatory function

6. Conclusion • The first enhancer region ranges from -80 to -11 relative to the transcription start site. • The second enhancer region ranges from +10 to +52. • The repressor element is located between position -11 and +8. • The first enhancer region contains several cis-acting elements. • The regulatory elements in the first enhancer region acts combinatorialy. • The loss of a single enhancer site from the first enhancer region renders the repressor functional. • Deletion of a bigger part of the first enhancer region along with the repressor element renders the second enhancer to carry out transcription with the help of a part of the first enhancer region from alternative transcription start site.

Transcriptional regulation of drosophila Nanos