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Dissertation Information for Suzanne R.L. Young

NAME:
- Suzanne R.L. Young

DEGREE:
- Ph.D.

DISCIPLINE:
- Molecular Biology

SCHOOL:
- Indiana University (USA) (2005)

ADVISORS:
- David G. Skalnik

COMMITTEE MEMBERS:
- Kristin Chun
- Matthew Grow
- Simon Rhodes
- Ronald C. Wek

MPACT Status: Incomplete - Not_Inspected

Title: CXXC finger protein 1 is important for hematopoiesis

Abstract: This dissertation characterizes the role of CXXC Finger Protein 1 (CFP-1) during hematopoietic differentiation. CFP-1, encoded by the CXXC1 gene, is a transcriptional activator that binds unmethylated CpG motifs and is ubiquitously expressed. Mice lacking CFP-1 exhibit an embryonic lethal phenotype (E3.5-E6). Murine embryonic stem cells lacking CFP-1 are viable but unable to differentiate and exhibit 60-80% decrease in cytosine methylation. The zebrafish homologue of CFP-1 is ∼70% similar to the mouse CFP-1 and is expressed in zebrafish embryos in all tissues. Approximately 80% of CFP-1 morpholino oligonucleotide ( zCXXC1 MO) treated embryos exhibited few circulating erythrocytes and abnormal yolk sac morphology at 48 hpf. More than half of the zCXXC1 MO treated zebrafish exhibited cardiac edema, and ∼14% were dead at 24 hpf. These phenotypes were rescued upon co-injection of the CFP-1 mRNA with the zCXXC1 MO. Embryos treated with a control MO exhibited normal yolk sac morphology and normal erythrocyte circulation. Genomic DNA from zCXXC1 MO treated embryos exhibited a 60% reduction of global cytosine methylation compared to DNA isolated from untreated or control MO treated embryos.

To examine the role CFP-1 during hematopoietic differentiation, RNA interference was used to inhibit CFP-1 expression in the PLB-985 myeloid leukemia cell line. Stable transfection of a short hairpin RNA (shRNA) targeted to CFP-1 resulted in 80% fewer drug resistant PLB-985 colonies compared to a vector control, suggesting that CFP-1 is required for PLB-985 cell survival. One stable colony exhibited a 70% decrease in CFP-1 protein levels, CFP-1 AS1. CFP-1 AS1 cells exhibited a slower doubling and 25% fewer cells in S phase. CFP-1 AS1 cells displayed a reduced ability to undergo granulocytic differentiation as ∼50% of the cells failed to obtain a terminally differentiated nuclear architecture and failed to generate a respiratory-burst. Similar results were obtained upon induction of monocyte/macrophage differentiation. Extended passaging of CFP-1 AS1 resulted in increased CFP-1 protein levels, ∼85% of wild type protein, and concomitant rescue of monocytic and granulocytic differentiation. Results from the zebrafish the RNAi studies demonstrate that CFP-1 is required for hematopoietic differentiation and establishes the importance of CFP-1 during post-gastrulation development.

MPACT Scores for Suzanne R.L. Young

A = 0
C = 0
A+C = 0
T = 0
G = 0
W = 0
TD = 0
TA = 0
calculated 2010-10-05 17:32:42

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