May be your promoter fragments are methylated after transfection. Try a methylation specific PCR to get answer for that.

No fragment showed any activity, but the positive control did, indicating that your experiment was properly done. I can be totally wrong, but I'd imagine that the fragments you cloned are not sufficient to promote transcription. Is there any possibility that additional DNA sequence (e.g., enhancer) that locates far from the promoter region is essential for the transcription?

Thanks Goodwin G. Jinesh for your answer, but how the cell line I'm using can methylate these fragments and it is expressing my protein. Also, how methylation specific PCR can be used to detect the methylation status of my promoter fragments after being transfected as it is non stable transfection?

Many thanks

Thanks Tomoyasu Sugiyama for your response. Actually my truncated fragments cover a region of 4 kb upstream of the TSS and also 5 kb downstream of the TSS. So I'm really surprised that another enhancing sequence could be present in a region farther than 5 kb downstream of the TSS, but if it do really exists how can I know this sequence.

What is the signal intensity you get of your empty vector? If it's very low than it is likely you get lower signal in your clones just because of different noise levels. If on the other hand you get a strong signal of the empty vector than you most likely have some contamination in your plasmid prep from another vector and you may be just normalizing your signal away. You may also want to check, especially in the constructs that extend downstream of the TSS if you haven't accidentally introduced a translation start site upstream of the Luc ATG resulting in an out of frame translation of the luciferase gene.

Methylation can be quick reaction, so it is still possible. Here is the link for methylation specific PCR

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC38513/

Enhancers can be far away from the 5kb limit you had mentioned. Check the promoter strength of your target sequence here

https://www-bimas.cit.nih.gov/molbio/proscan/

What substrate are you using for your NanoLuc? Have you sequenced your plasmids to confirm 1) the vector is correct and 2) your inserts are correct? How did you decide what direction to clone your cis-acting elements? Is it possible these are highly directional for transcription in the anti-sense orientation?

Check the transfection is ok, make sure the  vector  of fusgene is work 

For these type of assays, I always spike all my transfections with ~50 ng of a constitutive GFP expression plasmid to ensure there is no toxicity (bad prep) or transfection issues.

Dual luciferase - you are using the control vector and it is good? That also would indicate no transfection problems. I personally prefer NEB's secreted luciferases (GLuc & CLuc) much, much better than Promega's (which admittedly was state of the art when it was introduced). Secreted is more sensitive, easier (sample medium instead of lysate), more sensitive (at least GLuc is), and because it is secreted one can do a time course using a single well (single transfection).

Gut feeling - like many other RGers before me, the issue is with your cloned promoter fragment (it ain't what it's suppose to be). If the constructs really are exactly what you wanted, essential regulatory elements lie elsewhere.

Regarding Tomoyasu's very astute comment about a missing transcriptional regulatory sequence more than +/-5 kb away. My postdoc adviser's lab spent much more than a couple years to locate one more than 50 kb away from the transcribed gene (very thankfully before I arrived, but it was really a *very* important finding at the time that demonstrated how transcriptional enhancers can function at very great distances). Regulators for such genetic loci as the globin and hox families have since been shown to function much farther than even that (MBs). Open minds, and all that. But always, always balance risk and resource investment to best possible outcome (i.e. is it important to know what the regulatory regions of gene X are - sometimes YES and sometimes an emphatic no!) Looking for a missing transcriptional regulatory element can be a very lonely task without any guarantee of success. If you are in an academic track, *always* make sure you have a (side, if your primary is a bit risky) project that is virtually guaranteed to succeed and that will keep your spirits up so you can continue doing/enjoying research (and provide you substance for your future job talk!)

Also, definitely check out your gene on the University of California at Santa Cruz's Genome Browser. The annotations for several genome-wide characterizations such as histone modification hotspots (e.g. H3K27Ac) and transcriptional binding sites will clearly identify your most probable transcriptional regulatory regions. If you work on an organism that has not been characterized, check out the next closest species as regional functionality is usually conserved.

http://genome.ucsc.edu/cgi-bin/hgGateway

Thanks Craig M Neville for your help, According to NCBI the core promoter region is located inside one of my fragments. Also, conservation analysis among different species showed that 4 kb upstream the TSS is a highly conserved region so that's why I'm confused why my fragments aren't giving any activity

Thanks Daniel M Cohen for your feedback. I'm using ONE-Glo EX reagent to measure Firefly luciferase followed by incubation for 15 minutes. . Then NanoDLR Stop & GLo reagent was added to quench the Firefly luciferase signal and to provide furimazine substrate needed to measure NanoLuc signal and was incubated for 10 minutes. My gene is transcribed on the plus strand and according to the sequencing results my fragments are inserted in the right orientation upstream of luciferase gene.

Have you aligned the sequences to make sure there is nothing extra between your cloned DNA and the Nanoluc gene? Other than a bad transfection, there is no way that you can end up with lower activity than an empty vector unless you have accidentally introduced a transcriptional termination signal or made an ORF upstream and out of frame with the Nanoluc reporter. Try checking for Nanoluc mRNA by real-time PCR- this would resolve whether you are making mRNA that isn't getting translated or whether the low luciferase activity is truly because of a failure to induce transcription.

@Daniel M Cohen

One of my experimental vectors (having one of my fragments) is giving a signal of 2454 while the empty vector is giving a signal of 4171, this means that Nanoluc is translated in both but to a lower extent in my experimental vector, so if I performed real-time PCR I will get mRNA and a signal. But how can I differentiate between having a low signal due to frame shift mutation by the introduction of an ORF upstream of the reporter gene and having low signal because my fragments can't induce transcription

4171 sound a bit high for the empty vector, when using firefly and rennila luciferase I'm usually not getting reads of more than a couple of hundreds for the empty vectors.

the empty vector reporter gene is neither firefly nor renilla luciferase. It is Nanoluc which is a small enzyme (19.1kDa) engineered for optimal performance as a luminescent reporter. The enzyme is about 100-fold brighter than either firefly (Photinus pyralis) or Renilla reniformis luciferase using a novel substrate, furimazine, to produce high intensity, glow-type luminescence. So even the empty vector has intrinsic activity which is brighter than either firefly or renilla luciferase empty vectors.

If you try real-time PCR, make sure you perform DNAse I digestion of your nucleic acid or you will end up with a high amount of plasmid contamination in your mRNA. For real-time PCR, design primers against Nanoluc coding sequence, a housekeeping gene, and a non-transcribed region of your plasmid (negative control to check for plasmid contamination). You would expect to find the Nanoluc mRNA to be about 2x lower in your cells transfected with your experimental vector (a transcriptional effect). If instead you see much higher mRNA level in experimental vs empty vector control, then there has to be a problem with an upstream ORF.

Thanks a lot Daniel M Cohen, I will work on your suggestion and see whether I will find a leading result.

I can't tell from your posts whether your promoter constructs span the endogenous TSS or not. Enhancers will not give you decent luciferase expression unless you provide a TSS from a minimal promoter (sometimes included in the design of the vector, but I think it is not present in the vector you are using), or from your GOI. If no TSS is present, I would redesign the experimental vectors to include one in future iterations.

Actually all my truncated fragments contain the endogenous TSS sequence.