Research Interests

 

Our research program is focused on the eukaryotic DNA damage response and the etiology of hereditary cancer susceptibility syndromes associated with defective DNA repair. We study these fields through the lens of the rare genetic disease Fanconi anemia (FA). Therapeutic options for FA patients are limited and the overall life expectancy of FA patients is approximately 29 years. The molecular etiology of FA is poorly understood, and no rational therapeutic approaches based on the biochemistry of this disease have been developed. Progress in this field will only be achieved by a greater understanding of the molecular basis of this disease. The ultimate goal of our research program is to improve our understanding of the molecular basis of this disease to mitigate disease burden and extend patient lifespan.

The following is a summary of some current areas of research interest:

Fanconi Anemia and Targeted Transcriptional Regulation: Numerous studies have established a major role for the FA pathway in the repair of DNA interstrand crosslinks in vitro (Kottemann and Smogorzewska, 2013) . However, the endogenous – and physiologically relevant – source of genome instability in FA patients remains to be determined. Several studies have demonstrated an important role for the FA pathway in the mitigation of DNA replication stress. For example, we, and others, have shown that the FA proteins are required for the efficient replication of common chromosomal fragile sites (CFSs) and for the protection of stalled replication forks from degradation (Howlett et al., 2005; Madireddy et al., 2016; Schlacher et al., 2012) . Recent ChIP-seq data from our group and others has uncovered a remarkable nucleation of the FANCD2 protein at select large genes across the genome under conditions of replication stress (Okamoto et al., 2018) . Indeed, while ~1% of all human genes are >0.5 Mb, 70% of the genes that FANCD2 binds to under conditions of replication stress are >0.5 Mb. Large gene size and active transcription appear to be the primary determinants of FANCD2 binding. We propose that FANCD2 gene binding under conditions of replication stress may suppress transcription to prevent replication-transcription conflicts and prevent genome instability. We are currently interrogating this hypothesis using a targeted gene approach – focusing on qPCR and western blotting analysis of select FANCD2 gene targets – and by using RNA-seq and ATAC-seq analyses. We are also planning to conduct single-cell transcriptomics analysis of mutant and complemented FA patient cells to assess the level of transcriptional heterogeneity variation/noise at FANCD2 gene targets under conditions of replication stress.
A C. elegans Model of Fanconi Anemia Neurological Syndrome (FANS):  Although FA is primarily characterized by increased risk for bone marrow failure and cancer, several recent clinical reports describe pleiotropic neurological symptoms in FA patients, including abnormal brain MRIs, seizures, brain lesions, and early-onset cognitive decline. This constellation of neurological symptoms is collectively referred to as Fanconi Anemia Neurological Syndrome (FANS). The molecular etiology of FANS is unknown. Recent omics data from our lab and others suggests that the FANCD2 protein may play an important role in nervous system development under conditions of replication stress. For example, ChIP-seq analysis has uncovered that the FANCD2 protein binds to several transcriptionally active large neural genes upon treatment with the DNA polymerase inhibitor aphidicolin. Many of these large neural genes are linked to neuropsychiatric and neurodevelopmental disorders. Gene set enrichment analysis (GSEA) of RNA-seq data from FA-D2 (FANCD2-/-) and FANCD2-complemented patient cells has also uncovered differential regulation of neurogenesis and retinoic acid metabolism pathways in FA-D2 (FANCD2-/-) cells. We are currently using the model nematode Caenorhabditis elegans to study the role of the worm ortholog of FANCD2, FCD-2, in nervous system development. C. elegans has an exceptionally well-characterized nervous system with exactly 302 neurons, making it an excellent model for neuronal studies. conditions of replication stress. We anticipate that our findings will yield much needed insight into the origins of FANS and open new avenues of potential therapeutic intervention.
Fanconi Anemia, Retinoic Acid Signaling, and Retinaldehyde Genotoxicity: FA patients frequently exhibit heterogeneous and multisystemic congenital abnormalities. Patients from certain FA complementation groups are also at increased risk for embryonal tumors. These observations suggest defects in a key developmental biology pathway in FA and the existence of a FA-specific teratogen and embryonal mutagen. In recent RNA-seq studies, we have discovered dysregulation of retinoic acid biosynthesis and signaling in FA-D2 (FANCD2-/-) fetal fibroblasts. These findings are significant for two reasons, 1) retinoic acid signaling is critical for embryonic patterning, growth, and organogenesis and 2) retinaldehyde – an intermediate in retinoic acid biosynthesis – is a potent mutagen and teratogen. Our findings lead us to hypothesize that aberrant retinoic acid signaling and/or excess levels of the endogenous metabolite retinaldehyde may play a critical role in the pathogenesis of FA. We are currently testing this hypothesis using several approaches: 1) We are studying retinoic acid biosynthesis and signaling in transformed and non-transformed human and mouse FA cell types from several FA complementation groups. 2) We are also examining the functional implications of these findings: we are determining if FA patient cells exhibit increased sensitivity to retinoid cytotoxicity and genotoxicity. We are also currently examining if the differential expression of retinoic acid biosynthesis genes observed in FA fetal fibroblasts is a compensatory mechanism to mitigate the cytotoxic and genotoxic effects of endogenous retinoids. 3) We are also analyzing transcriptional regulatory targets of retinoic acid in FA. We are currently evaluating the differential expression of direct and indirect retinoic acid transcriptional targets in FA-D2 fetal fibroblasts, e.g., HOX, PAX, and SOX genes, using qPCR and immunoblotting. In summary, we have discovered a potentially important link between retinoic acid metabolism and FA. As retinoic acid metabolism is both nutritionally and therapeutically actionable, our studies could pave the way for improved dietary/prophylactic and therapeutic interventions for FA patients.
Fanconi Anemia and MiDAS: Several FA proteins have been functionally linked to the process of mitotic DNA synthesis, also known as MiDAS. While the bulk of DNA synthesis occurs during S-phase, under conditions of replication stress, e.g., following treatment with aphidicolin (APH), DNA synthesis can still be observed during prophase/prometaphase. This mitotic DNA synthesis process is thought to be required for the resolution of late replication intermediates to allow sister chromatid disjunction in anaphase (Garribba et al., 2018) . MiDAS resembles a homologous recombination process known as break-induced DNA replication (HR/BIR), which promotes the repair and restart of collapsed DNA replication forks. Important roles for POLD3, RAD52, FANCD2 and SLX4 in MiDAS have been established (Bhowmick et al., 2016; Graber-Feesl et al., 2019) . We are particularly interested in the roles of the FANCD2 and FANCI proteins in MiDAS and are using several complementary approaches to address this question. These studies will yield important insight into a poorly understood process and shed light on the physiological function of the FA proteins.
Determining the Role of the Fanconi Anemia Pathway in the Suppression of Structural Variation: Copy number variation (CNV) refers to genomic deletions or duplications of tens of thousands to millions of nucleotides. CNV is both a normal feature of genetic variation and a major contributor to genetic disease, e.g., neurological disease and cancer. The mechanisms by which CNVs arise and the cellular pathways that suppress CNV formation are largely unknown. We are currently collaborating with the Glover and Wilson groups in the Department of Human Genetics at the University of Michigan. Our collaborators have established that agents that perturb normal replication and create conditions of replication stress, e.g., aphidicolin, are potent inducers of nonrecurrent CNVs in cultured human cells (Arlt et al., 2009; Arlt et al., 2012; Wilson et al., 2015) . The Glover and Wilson labs have also established that active large transcription units (<1 Mb) drive extreme locus- and cell-type-specific genomic instability under replication stress resulting in CNV formation (Wilson et al., 2015) . We have compared the genome-wide distributions of replication stress-induced CNV hotspots to FANCD2 ChIP-seq peak regions and have established that there is considerable overlap. Furthermore, in preliminary experiments using both aCGH and SNP arrays, we have determined that the FANCA and FANCD2 proteins may play an important role in the suppression of de novo CNV. We are currently awaiting results of additional RNA-seq and ChIP-seq experiments with hTERT-immortalized mutant and complemented FA-D2 patient cells to select specific actively transcribed large transcription units for CNV analysis using customized genotyping arrays.

Bibliography

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2. Arlt, M.F., Wilson, T.E., and Glover, T.W. (2012). Replication stress and mechanisms of CNV formation. Curr Opin Genet Dev 22, 204-210.
3. Bhowmick, R., Minocherhomji, S., and Hickson, I.D. (2016). RAD52 facilitates mitotic DNA synthesis following replication stress. Mol Cell 64, 1117-1126.
4. Garribba, L., Wu, W., Özer, Ö., Bhowmick, R., Hickson, I.D., and Liu, Y. (2018). Inducing and Detecting Mitotic DNA Synthesis at Difficult-to-Replicate Loci. Methods Enzymol 601, 45-58.
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7. Kottemann, M.C., and Smogorzewska, A. (2013). Fanconi anaemia and the repair of Watson and Crick DNA crosslinks. Nature 493, 356-363.
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10. Schlacher, K., Wu, H., and Jasin, M. (2012). A distinct replication fork protection pathway connects Fanconi anemia tumor suppressors to RAD51-BRCA1/2. Cancer Cell 22, 106-116.
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