Grant: Discovery Grants Program - Individual

The ATRX chromatin remodeling protein is a key suppressor of genomic instability. My laboratory previously reported that ATRX depletion in human somatic cells leads to cell division defects and chromosomal abnormalities, and that loss of ATRX leads to DNA replication stress and cell death in the developing mouse brain. Moreover, ATRX is required for the normal expression of guanine-rich genes and the enzyme that makes an RNA copy from these genes (RNA PolII) accumulates in the gene bodies in ATRX-null cells, reducing the amount of RNA produced.***Genomic instability can be caused by the formation of structures called R-loops that often form at G-rich genes upon passage of RNA PolII. This can potentially cause ""fender-benders"" between the DNA replication and RNA transcriptional machineries, leading to DNA breaks and genome instability. Cellular factors are implicated in resolving R-loops but the mechanisms are presently poorly understood. We hypothesize that ATRX safeguards genomic stability by limiting the degree of R-loop burden in the cell nucleus.****Aim 1: Investigate whether ATRX limits the co-transcriptional formation of R loops***We will evaluate whether R-loops accumulate in the absence of ATRX in two different types of mouse cells and whether they coincide with RNA PolII accumulation. As a positive control, we will also deplete cells of the THO subunit Thoc1, which is known to accumulate R-loops. We will overexpress the enzyme RNAse H1, which degrades RNA in RNA-DNA hybrids and therefore resolves R-loops and should decrease their formation as well as DNA damage in ATRX-null cells.****Aim 2: Test whether DNA replication stress in ATRX-null cells is mediated by R loops***We propose that the formation of R-loops in G-rich gene bodies upon transcriptional elongation is the root cause of the replication stress, either because of collision of the replication and transcription complexes and/or to DNA hypercondensation around R-loop sites. The single molecule DNA fiber assay (molecular combing) will be used to directly measure DNA replication parameters via the dual-labeling of active replication forks. This assay detects replication fork speed, the density of active replication origins and the frequency of replication fork stalling. Overexpression of RNAse H1 will resolve which aspects of DNA replication defects result from the aberrant formation of R-loops in the absence of ATRX.***Importance of the work***The proposed studies will help us understand how R-loops influence gene expression and genomic instability. The research will further define how R-loops are maintained and eliminated in cells, and which proteins are involved in the regulation of these processes. Uncovering novel aspects of R-loop biology will shed light on many basic biological questions related to genomic instability and might instruct the development of synthetic probes or agents to detect, enhance or limit R-loops in cells in vivo.*** **