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Area of Expertise: Chromatin modifying complexes in Drosophila development
as a model for neurodegenerative disease and cancer
1. Histones were extracted from SAGA
mutant third instar larvae and probed with antibodies against histones H2B,
monoubiquitylated histone H2B (ubH2B), H3 and acetylated Lys-9 H3 (H3K9ac). The
H2B antibody also recognizes ubH2B (upper band). Mutations in nonstop and sgf11 result
in a global increase in ubH2B but do not affect acetylated Lys-9 H3 levels.
Conversely, mutation of the SAGA subunit ada2b reduces acetylated Lys-9
H3 but does not result in an increase in ubH2B (Weake et al. 2008 EMBO
2. In third instar larvae,
photoreceptor cells from the eye disc extend axons through the optic stalk (os)
into the optic lobe. The projection of R2 - R5 axons was visualized in wild type
and sgf11 larval optic lobes using the ro-tlacZ
marker (red). R1 – R6 axons terminate in the lamina (dotted lines) in wild type
within a triple layer of glial cells, visualized using anti-repo (green). In sgf11 optic
lobes, R2 – R5 axons project through the lamina into the medulla (me) and an
increased number of glial cells accumulate at the edges of the lamina
(arrowheads) while fewer glial cells are present along the lamina (Weake et
al. 2008 EMBO J.).
In eukaryotes, such as yeast, flies and humans, our DNA is compacted into a
nucleoprotein structure known as chromatin. The histone proteins that wrap
around the DNA to form chromatin can be modified by the addition of small
chemical and protein molecules, and these modifications are important for
regulating both gene expression and genomic integrity. In our lab, we study the
SAGA chromatin modifying complex using the fruitfly, Drosophila melanogaster,
as a model system. SAGA is a large multi-subunit complex and has two distinct
histone modifying activities. It is both a histone acetyltransferase and a
histone deubiquitylating enzyme. Intriguingly, the two activities of the complex
can be separated using mutations in different subunits. Mutations that disrupt
the histone acetyltransferase activity of SAGA such as ada2b reduce
global levels of acetylated Lys-9 on histone H3 (Figure 1). In contrast,
mutations that affect the histone deubiquitylase function of SAGA such as sgf11 or nonstop increase
global levels of monoubiquitylated histone H2B (Figure 1). However, mutations
in ada2b do not affect levels of monoubiquitylated histone H2B.
Furthermore, mutations in sgf11 or nonstop do not affect
acetylation of histone H3 at Lys-9. We are interested in understanding how the
different activities of the SAGA complex function mechanistically to regulate
transcription and gene expression in specific cell types. Misregulation of SAGA
subunits is associated with poor prognosis in specific types of cancer, and we
hypothesize that specific activities of SAGA are required in particular cell
types for proper regulation of gene expression and cell division. Why do
we think SAGA has tissue-specific functions? Our previous work has identified a
role for SAGA in regulating neuronal connectivity in the developing fly eye.
Mutations in sgf11 or nonstop disrupt targeting of photoreceptors
from the eye imaginal disc into the optic lobe (Figure 2). Previous studies
indicate that SAGA is required in glial cells rather than neurons for proper
photoreceptor axon targeting. We are interested in finding out why SAGA is
required for axon targeting by identifying genes that are regulated by SAGA in
glial cells, and by searching for potential non-histone targets of SAGA in the
brain. This work might provide insights into human neurodegenerative disease
because a hereditary human neurodegenerative disorder, spinocerebellar ataxia 7,
results from mutation of a SAGA subunit also involved in histone
deubiquitylation. Furthermore, this ataxia is associated with retinal
degeneration and blindness, suggesting that SAGA could also play an important
role in eye development and function in humans.
Awards & Honors
(2002) Massey Scholarship.
(2002) NZ Federation of Graduate Women Manawatu Branch Scholarship. Hildegard Gabriel Scholar (Predoctoral).
(2005) Top Achiever Doctoral Scholarship. Foundation for Research, Science & Technology.
(2010) Winner of Postdoctoral Oral Presentation. Stowers Institute for Medical Research, Crossroads Postdoctoral and Student Association Research Day..
Stephenson, R., Hosler, M., Gavande, N., Ghosh, A., & Weake, V. (2015). Characterization of a Drosophila ortholog of the Cdc7 kinase: A role for Cdc7 in endoreplication independent of Chiffon. J. Biol. Chem, doi:10.1074/jbc.M114.597948. Retrieved from http://www.jbc.org/content/290/3/1332.full.pdf+html
Weake, V. M., Ma, J., Brennan, K. J., D'Aloia, M. D., & Pascuzzi, P. (n.d.). SAGA deubiquitylase activity regulates glial migration through activating transcription of Rho and Multiplexin. Journal of Neuroscience.
Weake, V. M., Spreacker, P. J., Swanson, S., Florens, L., Washburn, M., & Stegeman, R. (n.d.). Spliceosomal proteins recruit SAGA to transcribed regions. Plos One.
Ma, J., & Weake, V. (2014). Affinity-based isolation of tagged nuclei from Drosophila tissues for gene expression analysis. J. Vis. Exp., 85, e51418. Retrieved from http://www.jove.com/pdf/51418/jove-protocol-51418-affinity-based-isolation-tagged-nuclei-from-drosophila-tissues-for
Mohan, R., Dialynas, G., Weake, V., Liu, J., Martin-Brown, S., Florens, L., Washburn, M., . . . Abmayr, S. (2014). Loss of Drosophila Ataxin-7, a SAGA subunit, reduces H2B ubiquitination and leads to neural and retinal degeneration. Genes Dev., 28, 259-272. Retrieved from http://genesdev.cshlp.org/content/28/3/259.long