David C. Zappulla
, Ph.D.




Department of Molecular Biology & Genetics
Johns Hopkins University - School of Medicine
725 North Wolfe Street, PCTB 620
Baltimore, MD 21205

zappulla[at]jhu.edu

office: (410) 955-3022
lab: (410) 516-8749
fax: (410) 955-0831
Current research:

My lab studies the RNP enzyme telomerase, telomeres, and senescence.


What is the physical organization of telomerase RNA and how does it relate to function?

I determined the secondary structure of the 1157-nt S. cerevisiae telomerase RNA while I was a postdoc with Tom Cech, and revealed that it functions as a flexible scaffold for the essential Est1 protein subunit. With NIH K99/R00 funding, my lab then demonstrated that this novel function for an RNA in an RNP complex also pertains to the other two S. cerevisiae telomerase holoenzyme-specific  subunits, Ku and Sm7, as well as for the essential three-way junction in the evolutionarily distant fission-yeast telomerase RNA. My lab has also unveiled several new essential well-conserved RNA features within yeast and human telomerase RNAs. The characteristics of certain new elements demonstrate that telomerase RNAs have functions that go beyond acting as flexible scaffolding for protein subunits. We are now determining the binding interface between the RNA and the catalytic protein subunit (TERT) at the core of the RNP enzyme, the extent of flexibility of telomerase RNA in the holoenzyme, as well as new telomerase subunits and their functions.






How is the telomerase RNP enzyme regulated to maintain telomere length?

Telomerase is preferentially recruited to short telomeres to promote their extension, but how this occurs is not known. We recently discovered how telomerase is recruited to telomeres by its Ku subunit. This finding led me to a model for the molecular mechanism of telomere-length homeostasis in yeast, as well as why there are two telomerase-recruitment pathways, coordinated by the Ku and Est1 subunits. With a new R01 supporting this project, we are now testing my model. One aim is to determine the arrangement of telomere proteins along chromosome ends to help learn how they “sense” telomere length to preferentially telomerase recruitment to short telomeres.





What are the molecular and cellular hallmarks of senescence caused by telomere loss?

Using RNA-seq, my group has determined the genome-wide transcriptional response of cells undergoing senescence due to erosion of telomeres, as well as the “survivors” that emerge subsequently. Several major findings have come from these RNA-seq data: (1) senescence has distinct phases, each with a signature of hundreds of differentially expressed genes; (2) senescence is characterized by upregulation of >100 novel lncRNAs; and (3) autophagy is one of many induced cellular pathways. We are now actively identifying the roles of differentially expressed proteins and RNAs to define and characterize the hallmarks of cellular senescence caused by telomere erosion.





Publications:

Preprints:

    Hass, E.P. and Zappulla, D.C. (2017) Repositioning the Sm-binding site in S. cerevisiae telomerase RNA reveals RNP organizational flexibility and Sm-directed    3'-end formation. bioRxiv doi: https://doi.org/10.1101/167361

       Hass, E.P. and Zappulla, D.C. (2017) A yeast two-hybrid system based onCRISPR-dCas9 for investigating RNA-protein interactions. bioRxiv doi: https://doi.org/10.1101/139600             

Peer-reviewed articles: 

Chen, H., Xue, J., Churikov, D., Hass, E.P., Lemon, L.D., Luciano, P., Bertuch, A.A., Zappulla, D.C., Geli, V., Wu, J., and Lei.M. Structural insights into yeast telomerase telomerase recruitment to telomeres. Cell (in press)

Niederer, R.O., Papadopoulos, N. and Zappulla, D.C. (2016) Identification of novel noncoding transcripts in telomerase-negative yeast using RNA-seq. Scientific Reports 6, 19376; doi: https://doi.org/10.1038/srep19376

Mefford, M.A. and Zappulla, D.C. (2016) Physical connectivity mapping by circular permutation of human telomerase RNAreveals new regions critical for activity and processivity. Molecular and Cellular Biology 36(2):251–261.

Hass, E.P. and Zappulla, D.C. (2015) The Ku subunit of telomerase binds Sir4 torecruit telomerase to lengthen telomeres in S. cerevisiae. eLIFE 4:e07750.   

    *Highlighted on journal cover

    *Johns Hopkins press release: http://hub.jhu.edu/2015/09/14/telomere-proteins-cancer-aging

Lebo, K.J., Niederer, R.O. and Zappulla, D.C. (2015) A second essential function of the Est1 arm of yeast telomerase RNA. RNA 21:862–876.

Niederer, R.O., and Zappulla, D.C. Refined secondary-structure models of the core of yeast and human telomerase RNAs directed by SHAPE. RNA 21:254–261.

Mefford, M.A., Rafiq, Q., and Zappulla D.C.  RNA connectivity requirements between conserved elements in the core of the yeast telomerase RNP. (2013) EMBO Journal 13:32(22): 2980–2993.

Lebo K.J. and Zappulla D.C.  Stiffened yeast telomerase RNA supports function in vitro and in vivo. (2012) RNA 18: 1666–1678.

Zappulla D.C., Goodrich K.J., Arthur J.R., Gurski L.A., Denham E.M., Stellwagen A.E. and Cech T.R. (2011) Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP.  RNA 17:298–311. (corresponding author)

Zappulla D.C.*, Roberts J.N., Goodrich K., Cech T.R. and Wuttke D.S.* (2009) Inhibition of yeast telomerase action by the telomeric ssDNA-binding protein, Cdc13p. (*co-corresponding author) Nucleic Acids Research 37:354–367.

Box J.A., Bunch J.T., Zappulla, D.C., Glynn E.F., and Baumann P. (2008) A flexible template boundary element in the RNA subunit of fission yeast telomerase. Journal of Biological Chemistry 283(35):24224-33

Zappulla D.C. and Cech, T.R. (2006) RNA as a flexible scaffold for proteins: yeast telomerase and beyond. Cold Spring Harbor Symposia on Quantitative Biology (Symposium 71: Regulatory RNAs) 71:217–224.

Zappulla D.C., Maharaj A.M., Connelly J., Jockusch R., and Sternglanz R. (2006) Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs. BMC Molecular Biology 4: 40-72.

Zappulla D.C., Goodrich K., and Cech T.R. (2005) A miniature yeast telomerase RNA functions in vivo and reconstitutes activity in vitro. Nature Structural and Molecular Biology 12(12):1072-1077.

Zappulla D.C. and Cech, T.R. (2004) Yeast telomerase RNA: a flexible scaffold for protein subunits. Proceedings of the National Academy of Sciences 101(27): 10024-10029.

Andrulis E.D., Zappulla D.C., Alexieva-Botcheva, K., Evangelista, C. and Sternglanz, R. (2004) Targeted silencing screens at HMR identify novel transcriptional silencing factors. Genetics 166:631-635.

Zappulla D.C., Sternglanz, R., and Leatherwood, J. (2002) Control of DNA replication timing by a transcriptional silencer. Current Biology 12: 869-875.

Andrulis E.D.*, Zappulla D.C.*, Ansari A.*, Perrod S., Laiosa C.V., Gartenberg M.R., and Sternglanz, R. (* equal contribution)  (2002) Esc1, a nuclear periphery protein required for Sir4-based plasmid anchoring and partitioning. Molecular and Cellular Biology 22(23): 8292-8301.

Xie W., Gai X., Zhu Y., Zappulla D.C., Sternglanz R., and Voytas, D. (2001) Targeting of the yeast Ty5 retrotransposon to silent chromatin is mediated by interactions between integrase and Sir4p. Molecular and Cellular Biology

Andrulis E.D., Neiman A.M., Zappulla D.C., and Sternglanz R. (1998) Perinuclear localization of chromatin facilitates transcriptional silencing. Nature 394: 592-595.

Ong B.C., Zimmerman A.A., Zappulla D.C., Neufeld E.J., and Burrows F.A. (1998) Prevalence of factor VLeiden in a population of patients with congenital heart disease. Canadian Journal of Anesthesia 45(12): 1176-1180.

Tufarelli C., Fujiwara Y., Zappulla D.C., and Neufeld E.J. (1998) Hair defects and pup loss in mice with targeted deletion of the first cut repeat domain of the Cux/CDP homeoprotein gene.  Developmental Biology 200(1): 69-81.

Yandava C.N., Zappulla D.C., Korf B.R., and Neufeld E.J. (1996) ARMS test for diagnosis of factor VLeiden mutation, a common cause of inherited thrombotic tendency.  Journal of Clinical Laboratory Analysis 10(6): 414-417.
 

Grants & Awards:

NIH R01 grant from NIGMS (2017–2022)

March of DimesBasil O'Connor Starter Scholar Research Award



Interested in joining my lab? Please email me at zappulla[at]jhu.edu


Postdoctoral research: 

Reconstitution of yeast telomerase activity via modeling and miniaturizing yeast telomerase RNA 

During the beginning of my postdoc with Tom Cech (HHMI, CU Boulder), I derived the secondary structure of the 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, based on phylogenetics and RNA folding software predictions (Zappulla and Cech, 2004). Using this model as a guide, I then designed yeast telomerase RNAs that are as little as one-third the size of wild type, even smaller than human telomerase RNA (451 nt), yet nevertheless retained essential function in vivo (Zappulla et al., 2005). Furthermore, these miniaturized (Mini-T) telomerase RNAs allowed me to reconstitute yeast telomerase activity in vitro, something that was presumably not possible because of the wild-type RNA's massive size. 

The identification of reconstituted yeast telomerase activity allowed telomerase and telomere protein functions to be individually assessed for the first time. In collaboration with Debbie Wuttke's lab, we determined that purified telomeric DNA end-binding protein Cdc13 inhibits reconstituted telomerase activity (Zappulla et al., 2009), suggesting a pivotal role for this protein in regulating telomerase access in vivo


Yeast telomerase RNA secondary structure and its reduction to create "Mini-T." The model on the left is my model for the wild-type 1157-nt TLC1 RNA and I designed the 500-nt Mini-T RNA by assembling predicted functional domains (boxed), discarding more than half of the RNA while retaining function in vivo and allowing reconstituted activity for the first time in vitro.





Yeast telomerase RNA as a flexible scaffold

In addition to demonstrating that >70% of TLC1 RNA is dispensable for function in vivo and that the bulk of the sequence is also evolving rapidly (even among species of Saccharomyces), I have shown that the essential Est1 protein binding site in TLC1 RNA can be dramatically repositioned in the RNA with retention of function (Zappulla and Cech, 2004). Together, these results provide evidence that TLC1 RNA serves as a flexible scaffold for proteins in this RNP enzyme. Thus, the telomerase RNP is apparently quite different from, for example, the ribosome and is potentially better fit to a "beads" (proteins) on a "string" (RNA) model for its global architecture (for review, see Zappulla and Cech, 2006). 

 

FIGURE: Schematic of yeast telomerase RNA TLC1 secondary structure model bound to proteins (drawn approximately to scale). 
TLC1 RNA nucleotides (circles) are shown in rainbow spectrum colors (red ––> violet) correlating with the predicted accuracy of their modeling by Mfold RNA secondary structure prediction software (red = best-determined; Zuker and Jacobson, 1998). Telomerase RNA-binding proteins Est2p (TERT), Est1p, Ku heterodimer and Sm7 heteroheptamer are drawn to scale with the RNA. 

Of course, the discrete catalytic center of the RNP, where TLC1 binds the protein reverse transcriptase subunit (Est2) and telomeric DNA, is almost certainly structurally ordered for coordinating reverse transcription. Scaffold roles for RNAs may be quite prevalent in biology, applying to other RNPs in addition to yeast telomerase. Examples of other RNA scaffolds may include Xist RNA, certain viral RNAs (e.g. some IRES's), tmRNA, pre-mRNA/protein complexes, and others (Zappulla and Cech, 2006).  

 

I am now collaborating with Peter Baumann (Stowers Institute for Medical Research) on the fission yeast telomerase RNA secondary structure. This RNA, which is evolutionarily very distant, also has many hallmarks of being a flexible scaffold: it is very large (1213 nts), is evolving very rapidly, and preliminary modeling shows that it has long quasi-helical arms, much like TLC1. The flexibility even extends into the catalytic action of this RNP, as we have already discovered (Box et al, 2008).


PhD research: 

Transcriptional silencing, DNA replication and the nuclear periphery

While pursuing my PhD in the laboratory of Rolf Sternglanz, at Stony Brook University, I studied transcriptional silencing in yeast, as well as its relationship to timing of DNA replication origin firing. I demonstrated that an early origin of DNA replication in a euchromatic region of the genome could be reprogrammed to fire late by introducing a transcriptional silencing element, the HMR-E silencer, nearby (Zappulla et al., 2002). Tethering an early origin to the nuclear periphery, where silenced late-replicating chromatin resides, was insufficient to delay replication timing in the absence of forming silent chromatin at the locus, suggesting that perinuclear localization per se is insufficient for late replication timing. Strikingly, simply targeting of the silencing protein Sir4 to the early origin reprogrammed it to fire late while also repressing transcription of a nearby gene, further arguing that chromatin state was important for origin firing.  

I also investigated the biological function of Esc1 and Esc4/Rtt107 proteins, both of which were identified in the Sternglanz lab as having the ability to recruit the Sir2/3/4 silencing protein complex when artificially tethered to a chromosome (using the Gal4 system). I discovered that Esc1 localizes to the nuclear periphery and is required for membrane protein targeted silencing (see Andrulis et al., 1998 for identification of membrane protein silencing), and together we found that Esc1 binds Sir4 and is required for Sir4-based plasmid partitioining and anchoring in the nucleus (Andrulis/Zappulla/Ansari et al., 2002 -- N.B. co-first authorship).  Esc1 appears to serve nuclear lamina-type functions in budding yeast. As for Esc4, I did phylogenetic work to demonstrate that this protein has six BRCT motifs (not just four), binds Slx4 with the N-terminal four BRCTs, is important for DNA repair and that it also binds to Sir3 (using C-terminal two BRCTs), explaining why it causes targeted silencing (Zappulla et al., 2006). 


Other links:

Johns Hopkins seminars: Johns Hopkins Scientific Calendar (JHMI)

Yeast-related: Saccharomyces Genome Database (SGD)

Johns Hopkins Institute for Data-Intensive Engineering and Science (IDIES)

NIH Pathway to Independence K99/R00 Award Website

Tom Cech's website at University of Colorado at Boulder in Department of Chemistry and Biochemistry

    President and Investigator, Howard Hughes Medical Insititute (HHMI)

My PhD thesis advisor, Rolf Sternglanz, at Stony Brook University

Ellis J. Neufeld's laboratory at Harvard Medical School (Children's Hospital) where I was a technician for two years after graduating from Middlebury College. 

Protocols from: the Gottschling lab, Botstein lab

Mfold RNA folding prediction server  

RNAalifold RNA sequence alignment-based folding server

Other places where I have studied: 

    Middlebury College, Middlebury, Vermont

    Sea Education Association, Woods Hole, Massachusetts.  

    Rocky Mountain Biological Laboratory, Gothic, Colorado.

    The Center for Circumpolar Studies, Wolcott, Vermont. 

Extracurricular: Ultimate frisbee, golf, hiking, squash, sailing







Last updated: 11/13/2017