1. (Graduate School) Genomic instability, DNA repair and epigenetic regulation in Spermatogenesis: My graduate research was based in a lab well established and equipped to study both male and female reproduction questions. As neither germline can be cultured, all this work was done in vivo, which provided me with the consideration to view biological questions as they relate to the system or animal while still focusing my work on exact and precise biological contexts. These interests involved the resolution of DNA repair events and how errors in this process lead to various genomic abnormalities. My efforts helped determine the details behind an essential component for embryonic development of gametogenesis, namely BTBD12 and its role in Double Strand Break (DSB) repair and interaction with the kinase ATM. We found that the loss of BTBD12 inhibited primordial germ cell proliferation as well as increased apoptosis, rendering the developing male infertile due to massively depleted germ cells reserves. Rare spermatocytes that could survive displayed significant chromosomal abnormalities, consistent with improper deployment of proper DNA repair pathways, implicating BTBD12 in coordinating DNA repair processed in meiosis.
   Through collaboration, I was able to explore my interest in the poorly understood levels of epigenetic regulation of transcription that is active in the germline. This work uncovered the relationship between the polycomb group protein SFMBT1 protein with LSD1-CoREST in a repressive complex and how this complex works to occupy RNA Polymerase II binding sites, including those of replication dependent histones, in a cell cycle dependent manner. My contribution helped to demonstrate this relationship in vivo spermatogonia, both cytologically and through direct molecular interaction. This work ultimately showed that this complex and interplay with RNA Polymerase II is highly regulated and essential for meiotic progression.
   52. Sun X, Brieño-Enríquez MA, Cornelius A, Modzelewski AJ, Maley TT, Campbell-Peterson KM, Holloway JK, Cohen PE. FancJ (Brip1) loss-of-function allele results in spermatogonial cell depletion during embryogenesis and altered processing of crossover sites during meiotic prophase I in mice. Chromosoma. 2016 Jun;125(2):237-52. PubMed Central PMCID: PMC5415080.
   53. Zhang J, Bonasio R, Strino F, Kluger Y, Holloway JK, Modzelewski AJ, Cohen PE, Reinberg D. SFMBT1 functions with LSD1 to regulate expression of canonical histone genes and chromatin-related factors. Genes Dev. 2013 Apr 1;27(7):749-66. PubMed Central PMCID: PMC3639416.
   54. Holloway JK, Mohan S, Balmus G, Sun X, Modzelewski A, Borst PL, Freire R, Weiss RS, Cohen PE. Mammalian BTBD12 (SLX4) protects against genomic instability during mammalian spermatogenesis. PLoS Genet. 2011 Jun;7(6):e1002094. PubMed Central PMCID: PMC3107204.
2. (Graduate School) The role of small RNAs in transcriptional Regulation of Mammalian Meiosis My major graduate research contributions focused the largely overlooked, albeit critical, role of RNAi in the mammalian germ line. I demonstrated that AGO4 localizes to the normally silent XY bivalent (sex body), and any autosomes that remain asynapsed as a result of translocations or recombination errors, suggesting a role for AGO4 in silencing at regions of unsynapsed DNA. This finding is novel and surprising given the expected solely cytoplasmic role/location for RNAi machinery in mammals. While nuclear RNAi had only been speculated to exist, my findings provided the first direct functional evidence of this in vivo. This work was published in Development Cell in August 2012 and chosen as the article of the month as well as given the privilege of being freely accessible. As a follow up, I performed a parallel analysis on germ line conditional deletions of RNAi components essential for small RNA biogenesis: Dicer (miRNA and siRNA) and Dgcr8 (miRNA only), which was accepted in the Journal of Cell Science, revealing critical details behind this new mechanism. Although the mechanism behind the involvement of Ago4 and small RNAs in meiotic nuclear silencing is still not fully understood, this very question has generated multiple projects in the lab of my PhD advisor, Paula Cohen. Since the original manuscript, the concept of nuclear RNAi in mammalian systems has been slowly gaining traction. Since publication, this work was cited in a very positive light 25 times, demonstrating that the field is becoming more receptive to the prospect of this novel mechanism. The underlying implication that this work suggests is a completely new paradigm in our knowledge of transcriptional regulation, as it applies to potentially all biological processes in the mammalian system.
   235. Hilz S, Fogarty EA, Modzelewski AJ, Cohen PE, Grimson A. Transcriptome profiling of the developing male germ line identifies the miR-29 family as a global regulator during meiosis. RNA Biol. 2017 Feb;14(2):219-235. PubMed Central PMCID: PMC5324742.
   236. Hilz S, Modzelewski AJ, Cohen PE, Grimson A. The roles of microRNAs and siRNAs in mammalian spermatogenesis. Development. 2016 Sep 1;143(17):3061-73. PubMed Central PMCID: PMC5047671.
   237. Modzelewski AJ, Hilz S, Crate EA, Schweidenback CT, Fogarty EA, Grenier JK, Freire R, Cohen PE, Grimson A. Dgcr8 and Dicer are essential for sex chromosome integrity during meiosis in males. J Cell Sci. 2015 Jun 15;128(12):2314-27. PubMed Central PMCID: PMC4487015.
   238. Modzelewski AJ, Holmes RJ, Hilz S, Grimson A, Cohen PE. AGO4 regulates entry into meiosis and influences silencing of sex chromosomes in the male mouse germline. Dev Cell. 2012 Aug 14;23(2):251-64. PubMed Central PMCID: PMC3470808.
3. (Postdoc and Current) The role of Retrotransposon Reactivation in Mammalian Embryonic Development: My Post-Doctoral Fellowship research contribution actively focus on the role of Retrotransposon Reactivation in the settings of pre-implantation development. Previous research into transposable elements have assigned a parasitic and opportunistic role as the standard relationship between host and invader. I feel that this is an oversimplification, and through millions of years of co-evolution, the host genome has developed various mechanisms to domesticate transposable elements, which make up nearly half of mammalian genomes. Recent publications have reported interesting observations between retrotransposons and nearby gene expression; however, these have mostly been thought to be the result of disruptions in the mechanisms that typically hold these sequences in a silenced status, such is the case in various pathologies, including cancer. A hallmark of preimplantation development is the reactivation of retrotransposons, which is thought to be one of the few opportunities for these sequences to reproduce. However, we have found evidence that this reactivation is surprisingly coordinated and perhaps not a stochastic lapse in regulation, and instead, these reactivations are so precise their expression profiles can be used as a signature for each embryonic stage. As retrotransposons exist as dozens to thousands of copies of nearly identical sequences, each of the over 100 families are regulated in distinct ways, and presumably in a sequence dependent manner. Depending on proximity and orientation to nearby genes, the retrotransposons appear to elicit specific and potent effects on nearby genes expression, which can be predicted by the expression of the adjacent retrotransposon. Additionally, we have found hundreds of examples of retrotransposon-gene fusion events that modify the structure of the gene, and in some cases, truncate or augment the open reading frame, potentially generating stage specific protein isoforms of key developmental genes that have not been seen in any other cell type, except perhaps cancer. To this end, I published the very first report of a developmentally essential retrotransposon, where deletion of a single element results in profound peri0implnatation defects resulting in embryonic and maternal death. We believe we have uncovered a novel regulatory mechanism that normally occurs, and largely directs, pre-implantation development in both mouse and human. The important implication presented here highlights the unique property of retrotransposons to exist in multiple loci while having the nearly identical regulatory sequences, and it is through this similarity that genome wide coordinated expression can be achieved by regulating relatively few sequences, as the retrotransposons are silently poised nearby and inside genes across all chromosomes to act in a precisely coordinated manner. This hidden embryonic transcriptome is just now possible to be explored and will quickly pose many unanswered questions dealing with surprisingly similar events that occur in tumorigenesis.
   70022. Li TD, Toohill K, Modzelewski AJ. From Junk DNA to Genomic Treasure: Impacts of Transposable Element DNA, RNA, and Protein in Mammalian Development and Disease. Wiley Interdiscip Rev RNA. 2025 Jul-Aug;16(4):e70022. doi: 10.1002/wrna.70022. PMID: 40804709; PMCID: PMC12350819.
   70023. Guo Y, Li TD, Modzelewski AJ, Siomi H. Retrotransposon renaissance in early embryos. Trends Genet. 2024 Jan;40(1):39-51. PubMed PMID: 37949723.
   70024. Modzelewski AJ, Gan Chong J, Wang T, He L. Mammalian genome innovation through transposon domestication. Nat Cell Biol. 2022 Sep;24(9):1332-1340. PubMed Central PMCID: PMC9729749.
   70025. Modzelewski AJ, Shao W, Chen J, Lee A, Qi X, Noon M, Tjokro K, Sales G, Biton A, Anand A, Speed TP, Xuan Z, Wang T, Risso D, He L. A mouse-specific retrotransposon drives a conserved Cdk2ap1 isoform essential for development. Cell. 2021 Oct 28;184(22):5541-5558.e22. PubMed Central PMCID: PMC8787082.
4. (Postdoc and Current) CRISPR Cas9 mediated Genome Editing in Mammalian Embryos: During my primary Post-Doctoral Fellowship project, I was challenged with the need to test the role of retrotransposons activity in the pre-implantation mouse embryo. Although CRISPR/Cas9 technology has forever transformed mouse genome editing, the current practice of microinjecting CRISPR reagents into pronuclear-stage embryos remains expensive and rate-limiting. As no other cell system exists that can serve as a proxy for the embryo, all testing, optimizing, experiments and manipulations must be done in the embryo. To test the various novel regulatory systems discovered in our analysis, a massive effort would be needed that would largely rely on the schedule and fees of a skilled microinjection technician. As this was not only time prohibitive, but it was also massively cost prohibitive. To bypass this limitation entirely, I developed CRISRP-EZ (CRISPR RNP Electroporation of Zygotes), an electroporation-based technology that outperforms microinjection in efficiency, simplicity and throughput at a fraction of the cost. In C57BL/6J and C57BL/6N mouse strains, CRISPR-EZ achieves 100% delivery of Cas9/sgRNA ribonucleoproteins (RNPs), facilitating indel mutations (insertions or deletions), exon deletions, point mutations, and small insertions. Since the first publication, we have further optimized the protocol by a side-by-side comparison in the high-throughput KnockOut Mouse Project (KOMP) pipeline wherein CRISPR-EZ consistently outperformed microinjection and in many cases outright replaced it. This protocol covers single guide RNA (sgRNA) synthesis, embryo collection, RNP electroporation, mouse generation, and genotyping strategies. Using CRISPR-EZ, a graduate-level researcher can validate editing in embryos and obtain genetically modified animals in 6 weeks at a significantly reduced cost. Altogether, CRISPR-EZ is a simple, economic, efficient, and high-throughput technology that is potentially applicable to other mouse strains and mammalian species, with current efforts aimed at increasing the size of knock-in templates and expanding to other placental mammals, for example, cow and dog. Ref 3 and 4 below represent collaborative efforts where difficult to generate mouse models were made using CRISPR-EZ.
   710864. Björkgren I, Chung DH, Mendoza S, Gabelev-Khasin L, Petersen NT, Modzelewski A, He L, Lishko PV. Alpha/Beta Hydrolase Domain-Containing Protein 2 Regulates the Rhythm of Follicular Maturation and Estrous Stages of the Female Reproductive Cycle. Front Cell Dev Biol. 2021;9:710864. PubMed Central PMCID: PMC8455887.
   710865. Diallo, C. K., Modzelewski, A. J†. Efficient Genome Editing of Mice by CRISPR Electroporation of Zygotes. J. Vis. Exp. (190), e64302, doi:10.3791/64302 (2022)
   710866. Modzelewski AJ, Chen S, Willis BJ, Lloyd KCK, Wood JA, He L. Efficient mouse genome engineering by CRISPR-EZ technology. Nat Protoc. 2018 Jun;13(6):1253-1274. PubMed Central PMCID: PMC6296855.
   710867. Chen S, Lee B, Lee AY, Modzelewski AJ, He L. Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes. J Biol Chem. 2016 Jul 8;291(28):14457-67. PubMed Central PMCID: PMC4938170.

Complete List of Published Work in MyBibliography:   http://www.ncbi.nlm.nih.gov/sites/myncbi/1faUwlLilsuQc/bibliography/40938938/public/?sort=date&direction=ascending