Katie Nguyen on hypertension and David Klein on pluripotency

Katie Nguyen, Brodsky Lab

A Screen for ROMK Gain-of-Function Mutations that may give rise to Hypertension

Hypertension affects 1 billion people worldwide and is the most common risk factor for many cardiovascular diseases. While genetic causes contribute to up to 50% of all cases, it is difficult to pinpoint the genetic cause of hypertension due to its heterogenic nature. One of the major regulators of hypertension are the salt-handling transporters in the kidney, including the renal outer medullary K+ (ROMK) channel. While loss-of-function (LOF) mutations in ROMK give rise to Bartter’s Syndrome Type II, heterozygous carriers of these same mutations are protected from hypertension. Therefore, my hypothesis is that a gain-of-function (GOF) mutation in ROMK could predispose people to hypertension. As GWAS often overlook rare trait variants and can’t predict GOF phenotypes, an experimental approach to identify GOF mutations in ROMK would not only help determine one of the genetic factors of hypertension, but also strengthen our understanding of the dynamics that underlie ROMK structure-function. Towards this end, we expressed ROMK in a Saccharomyces cerevisiae strain lacking two major K+ transporters, so growth is partially restored on low K+, and then screened for clones that better rescue growth on low K+. So far, I have identified 98 mutations that mapped to 38 loci in the ROMK gene. Preliminary analysis showed that these mutations have varying degrees of stability and conductance. Going forward, I am optimizing the screen and will use different approaches to elucidate the mechanisms by which these mutations acquired their GOF phenotypes.


David Klein, Hainer Lab

Determining the role of the essential histone chaperone FACT in maintenance of ES cell pluripotency

Embryonic stem (ES) cells carefully regulate the decision to undergo a transition to a particular cell type (differentiation) or to maintain their undifferentiated state (pluripotency). This cell fate decision is moderated by a litany of spatial, temporal, and signaling cues, transcription factors, and chromatin-organizing factors. At the transcriptional level, pluripotency is maintained by a specific set of transcription factors, including Oct4, Nanog, Sox2, KLF4, and c-MYC. While these factors are essential for stem cells to maintain their pluripotent state, factors that regulate these pluripotency factors remain incompletely characterized. The facilitates chromatin transcription (FACT) complex is an essential histone chaperone and is comprised of two subunits, Spt16 and SSPR1. FACT promotes H2A/H2b dimer exchange and has roles in transcription, DNA repair, recombination, and replication. FACT is expressed in both proliferating and non-proliferating cells but its role in cell fate decisions is uncharacterized. Using murine ES cells, I aim to determine the role of FACT in pluripotency. I have reduced FACT expression in ES cells at both the mRNA and protein levels and assayed changes to the transcriptional landscape through RTqPCR and RNA-seq. I found that FACT depletion disrupts ES cells’ pluripotent state by reducing the expression of key pluripotency factors. Concurrently, we observed altered cell morphology upon FACT depletion. I have begun genome-wide mapping of FACT chromatin binding to assess whether these changes in gene expression are direct or indirect. Future work will is aimed at determining the mechanism through which FACT is required for ES cell pluripotency. 

Friday, October 5th

A219B Langley Hall

12 PM


05 Oct 2018

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Graduate Student Presentations


A219B Langley Hall