Description of Research Expertise

Research Interests
This laboratory is devoted to understanding basic mechanisms of DNA repair and their impact on genome integrity, cancer etiology and response to targeted therapies. To investigate these interrelationships, we are devoted to elucidating BRCA1- and BRCA2- dependent homologous recombination mechanisms in breast and ovarian cancer, telomere length maintenance mechanisms that rely on a specialized form of homologous recombination, and DNA damage induced activation of immune responses to cancer. We utilize a myriad of approaches to investigate these areas, which include biochemistry, structural biology, cell biology, and genetically engineered mouse models.

Key words: BRCA1, BRCA2, DNA repair, Homologous Recombination, Telomeres, Epigenetics, Breast Cancer, Ovarian Cancer, cytokines, immune therapy.

Description of Research

Germline mutation of either the Breast Cancer 1 (BRCA1) or Breast Cancer 2 (BRCA2) genes greatly predisposes individuals to breast and ovarian epithelial cancers. Clinical BRCA1 and BRCA2 mutations render cells deficient in DNA damage checkpoint signaling and error-free mechanisms of DNA repair known as homologous recombination, strongly supporting a role for these activities in tumor suppression. Our research has contributed to the concept of a BRCA-centered breast and ovarian tumor suppressor network. BRCA1 forms several distinct DNA damage inducible 'supercomplexes, each dedicated to specific checkpoint and repair activities following genotoxic stress. This work revealed a molecular understanding for how BRCA1 recognizes DNA damage sites. An interaction between the BRCA1 BRCT domain and the RAP80 ubiquitin binding protein targets BRCA1 to K63-linked ubiquitin structures present at DNA damage sites. The RAP80 ubiquitin interaction motifs (UIMs) provide an ubiquitin recognition element to target the BRCA1 E3 ligase and a K63-ubiquitin specific deubiquitinating enzyme BRCC36 to DNA double strand breaks (Sobhian et al. Science 2007; Jiang et al. Genes Dev 2015; Zeqiraj et al. Mol Cell 2015; Jiang et a. JCB 2022). Cancer causing BRCA1 mutants fail to interact with RAP80 and consequently demonstrate inefficient recruitment to DNA damage sites. Our subsequent studies identified biallelic mutations in BRCA1 as a cause of a new Fanconi Anemia subtype, and received HUGO approval to designate BRCA1 as FANCS (Sawyer et al. Cancer Discov 2015; Domchek et al. Cancer Discov 2013). This syndrome is characterized by developmental abnormalities consistent with Fanconi Anemia along with extremely early onset breast and ovarian cancer. We demonstrated that histone H4 acetylation promotes BRCA1 displacement of 53BP1 on damaged chromatin (Tang et al. Nat Struct Mol Biol 2013) to enable homologous recombination. This work was the first to show that transcriptionally active chromatin regions preferentially use homologous recombination over nonhomologous end-joining repair mechanisms. Recently, we revealed the importance of PARylation dependent chromatin remodeling to viability of BRCA mutant cancer cells. This work showed the PAR binding SNF2 ATPase, ALC1 is essential for base damage repair (Verma Nat Cell Biol 2021). Importantly, loss of ALC1 creates profound PARP inhibitor sensitivity in homologous recombination deficient cells and overcomes known resistance mechanisms, defining ALC1 as an exciting new drug target.

Telomere length maintenance is a requisite feature of cellular immortalization and a hallmark of cancer. Approximately 85% of cancers rely on the re-expression of telomerase reverse transcriptase, while nearly 15% utilize a recombination-based mechanism known as alternative lengthening of telomeres (ALT). We developed a methodology for real-time visualization of each major step of homology-directed DNA repair during ALT (Cho et al. Cell 2014; Dilley et al. Nature 2016, Verma et al. Genes Dev 2019; Jiang et al. Mol Cell 2024). Telomere damage initiated rapid directional ALT telomere movement, culminating in synapsis and homologous recombination dependent long tract telomere synthesis. These findings have implications for understanding large-scale chromatin dynamics, fundamental mechanisms of homology searches, and potential targets to selectively inhibit telomere maintenance in ALT positive cancers. We are actively pursuing these areas with the aspirational goal of defining the entire ALT process.

Our investigations in the DNA damage response have expanded to the question of how DNA damage activates innate immune responses. This revealed the importance of progression through mitosis and pattern recognition within micronuclei to DNA damage as critical steps in eliciting anti-tumor immune responses (Harding et al. Nature 2017; Chen et al. Cell Reports 2020; Lippert and Greenberg JCI 2021). We also described parallel ubiquitin-dependent signaling mechanisms between DNA damage and immune responses (Zheng et al. Cell Rep 2013; Zeqiraj et al. Mol Cell 2015; Walden et al. Nature 2019). Collectively, our research in this area provides new avenues for combining DNA damaging therapies with immune checkpoint blockade and insights into indications such as cancer and autoimmunity. We are pursuing these areas with the recent development of homologous recombination deficient ovarian cancer models that reveal new aspects of innate immune signaling that influences tumor progression and response top immune therapy.

Our interests in BRCA and telomere maintenance has inspired our investigations into the complex relationship between chromatin structure and genome integrity. We recently reported a protein complex that we named 55LCC that unfolds DNA replication factors to prevent replication stress and severe chromosome instability due to mitotic errors (Krishnamoorthy et al. Cell 2024). All four members of the 55LCC complex are mutated in a human neurodevelopment syndrome EHLMRS (Epilepsy, Hearing Loss, Mental Retardation Syndrome). This reveals a new aspect of genome integrity that we will continue to investigate. We have also developed the first systems to visualize DSB responses and nascent transcription in real time in human cells. This enabled our discovery of ATM dependent transcriptional silencing that extends kilobases from the site of DNA damage (Shanbhag et al. Cell 2010; Harding et al. Cell Reports 2015), which relates to myriad aspects of biology, including suppression of chromosome translocations, intrinsic responses to viruses, ane meiotic sex chromosome inactivation.



Rotation Projects
Rotation projects are open to students in each of the areas the lab focuses on. We are particularly proud of the seven students that have successfully completed PhD studies in our lab. Three Greenberg lab students have received the Saul Winegrad Award for best thesis and almost all graduating students have continued in research as postdocs in outstanding laboratories.

Greenberg Lab Website
Learn more about the lab's research here.

Penn Center for Genome Integrity Website
https://www.med.upenn.edu/pcgi/


Lab personnel:
Weihua Li - Research Specialist, Lab Manager
Adel Atari - Graduate Student
Taku Harada - Graduate Student
Xiaodi Chen - Graduate Student
Arindam Datta - Postdoctoral Researcher
Reona Kato - Postdoctoral Researcher
Stefano Misino - Postdoctoral Researcher
Jie Chen - Postdoctoral Researcher
Haoyang Jiang - Postdoctoral Researcher
Sangin Kim - Postdoctoral Researcher
Tao Shi - Postdoctoral Researcher
Kumari Prerna - Postdoctoral Researcher
Holly Qiu - Undergraduate Student
Aravind Krishan - Undergraduate Student



Administrative Coordinator:
Laura Murillo
215-573-0908
murillo@upenn.edu