Hadeel Ali

Green Fellows project abstract:

Poly(ADPribosyl)ation (PARylation) is a reversible post-translational modification that includes the covalent attachment of ADP-ribose moieties to proteins by poly(ADP-ribose) polymerases (PARPs), primarily PARP1. Previous work from the lab has shown PARP1 driven PARylation of C/EBPβ prevents binding to DNA thus it inhibits the expression of adipogenic genes. In macrophages, C/EBPβ is a lineage determining transcription factor (LDTF) that binds to chromatin and helps differentiate an unstimulated macrophage into a pro-inflammatory macrophage and drives pro-inflammatory gene expression. Therefore, we ask whether PARP1 mediated PARylation of C/EBPβ occurs in macrophages and what role it plays in pro-inflammatory gene expression. To investigate the role of PARylation of C/EBPβ regulatory domain sites E135 and E139, we mutated two glutamic acid sites of C/EBPβ’s regulatory domain to alanine so PARP1 can no longer ADP-ribosylate C/EBPβ. We made a DOX inducible system in immortalized bone marrow derived macrophages (iBMDM) to over express flag-tagged C/EBPβ. We verified generation of inducible ectopically expressed C/EBPβ (wild type “WT” and mutant “MUT”) within iBMDM by Western blot. Using fluorescent antibodies in a Western blot, we observed that there is a loss of PARylation on the C/EBPβ mutant as compared to the WT. This confirms that these sites are PARylated in macrophages. In order to determine the effect of loss of C/EBPβ PARylation on pro-inflammatory gene expression, we treated cells with bacterial Lipopolysaccharides (LPS) to induce inflammation. The expression of pro-inflammatory genes was analyzed using RT-qPCR. Macrophages expressing Mut C/EBPβ had lower pro-inflammatory gene expression than WT C/EBPβ. This shows PARylation of C/EBPβ occurs and is important for the expression of pro-inflammatory cytokines in macrophages. Our studies show the potential for targeting C/EBPβ PARylation in regulating unchecked inflammation in inflammatory and autoimmune diseases. 

 

​​What does research mean to you? 

Research means to me the endless pursuit of answering your questions. There is always more to know, more to find out, more questions that arise than answers. 

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Tell us about your journey​!

I entered undergrad undecided and fell in love with neuroscience after one class. I was new to the sciences in general, I didn't think it was something I wanted to pursue before entering university, but I've proved that wrong to myself now. I felt overwhelmed with all the new career options in front of me I now had to consider, and research was on top of the discovery list. 

From the end of freshman year to middle of sophomore year I worked in a UTD dry lab. I enjoyed the idea of contributing to a bigger project and wanted to expand the chance to work in the biological sciences research.

I learned about the Green Fellowship walking through the halls and seeing previous Fellow's posters. I knew I wanted to be up there and experience what they learned. I knew it was an invaluable opportunity to experience full time research, to see a glimpse of what a career in research is like.

I entered a molecular biology lab at UTSW and learned so many new techniques and approaches to answering questions. After the program I am still figuring out what exactly I want to pursue: MD, PhD, MD/PhD, and I carry the information I learned with me to make a more confident decision. 

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What was your favorite part about the program? 

I enjoyed getting to know the graduate students, post docs, and doctoral fellows in my lab. It was great hearing so many varied perspectives within their pursuit of scientific interest. It was interesting to hear the directions they encouraged me to go in. It was wonderful having a supportive growth minded environment. There was always someone to help me learn from my mistakes and answer my questions. It was great making friendships within the lab, having lunch, and laughing together. 

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What was the biggest thing you learned from the program?

I learned to not underestimate myself and respect the work I need to do to succeed. I felt so out of place working in a biology lab with no previous experience. I was self-conscious of my mistakes and felt paralyzed in how to approach learning so much new information at once. As the weeks went by, my confidence grew, and I knew that if I wanted to excel and take advantage of this opportunity, I needed to be fine outside of my comfort zone. I recognize the difference in the way I spoke about my project from its initial formation to the end. I am proud of that transformation. I now carry the confidence that even when I do not know something initially, I can learn.

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Advice for Future Green Fellows

It is okay to now know what you want to do or know what is happening all the time. The point is to put in the effort to do better and learn. Do not be the one to say no to yourself and hold yourself back. Go for it!

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