Molecular underpinnings and environmental drivers of loss of heterozygosity in Drosophila intestinal stem cells.

During development and aging, genome mutation leading to loss of heterozygosity (LOH) can uncover recessive phenotypes within tissue compartments. This phenomenon occurs in normal human tissues and is prevalent in pathological genetic conditions and cancers. While studies in yeast have defined DNA repair mechanisms that can promote LOH, the predominant pathways and environmental triggers in somatic tissues of multicellular organisms are not well understood. Here, we investigate mechanisms underlying LOH in intestinal stem cells in Drosophila. Infection with the pathogenic bacteria, Erwinia carotovora carotovora 15, but not Pseudomonas entomophila, increases LOH frequency. Using whole genome sequencing of somatic LOH events, we demonstrate that they arise primarily via mitotic recombination. Molecular features and genetic evidence argue against a break-induced replication mechanism and instead support cross-over via double Holliday junction-based repair. This study provides a mechanistic understanding of mitotic recombination, an important mediator of LOH, and its effects on stem cells in vivo.

Stem Cell DNA Damage and Genome Mutation in the Context of Aging and Cancer Initiation.

Adult stem cells fuel tissue homeostasis and regeneration through their unique ability to self-renew and differentiate into specialized cells. Thus, their DNA provides instructions that impact the tissue as a whole. Since DNA is not an inert molecule, but rather dynamic, interacting with a myriad of chemical and physical factors, it encounters damage from both endogenous and exogenous sources. Damage to DNA introduces deviations from its normal intact structure and, if left unrepaired, may result in a genetic mutation. In turn, mutant genomes of stem and progenitor cells are inherited in cells of the lineage, thus eroding the genetic information that maintains homeostasis of the somatic cell population. Errors arising in stem and progenitor cells will have a substantially larger impact on the tissue in which they reside than errors occurring in postmitotic differentiated cells. Therefore, maintaining the integrity of genomic DNA within our stem cells is essential to protect the instructions necessary for rebuilding healthy tissues during homeostatic renewal. In this review, we will first discuss DNA damage arising in stem cells and cell- and tissue-intrinsic mechanisms that protect against harmful effects of this damage. Secondly, we will examine how erroneous DNA repair and persistent DNA damage in stem and progenitor cells impact stem cells and tissues in the context of cancer initiation and aging. Finally, we will discuss the use of invertebrate and vertebrate model systems to address unanswered questions on the role that DNA damage and mutation may play in aging and precancerous conditions.

Spen limits intestinal stem cell self-renewal.

Precise regulation of stem cell self-renewal and differentiation properties is essential for tissue homeostasis. Using the adult Drosophila intestine to study molecular mechanisms controlling stem cell properties, we identify the gene split-ends (spen) in a genetic screen as a novel regulator of intestinal stem cell fate (ISC). Spen family genes encode conserved RNA recognition motif-containing proteins that are reported to have roles in RNA splicing and transcriptional regulation. We demonstrate that spen acts at multiple points in the ISC lineage with an ISC-intrinsic function in controlling early commitment events of the stem cells and functions in terminally differentiated cells to further limit the proliferation of ISCs. Using two-color cell sorting of stem cells and their daughters, we characterize spen-dependent changes in RNA abundance and exon usage and find potential key regulators downstream of spen. Our work identifies spen as an important regulator of adult stem cells in the Drosophila intestine, provides new insight to Spen-family protein functions, and may also shed light on Spen's mode of action in other developmental contexts.