Faculty Bibliography

When an essential amino acid is limited, a signaling cascade is triggered that leads to increased translation of the 'master regulator', activating transcription factor 4 (ATF4), and resulting in the induction of specific target genes. Binding of ATF4 to the amino acid response element (AARE) is an essential step in the transcriptional activation of CHOP (a CCAAT/enhancer-binding protein-related gene) by amino acid deprivation. We set out to identify proteins that interact with ATF4 and that play a role in the transcriptional activation of CHOP. Using a tandem affinity purification (TAP) tag approach, we identified p300/CBP-associated factor (PCAF) as a novel interaction partner of ATF4 in leucine-starved cells. We show that the N-terminal region of ATF4 is required for a direct interaction with PCAF and demonstrate that PCAF is involved in the full transcriptional response of CHOP by amino acid starvation. Chromatin immunoprecipitation analysis revealed that PCAF is engaged on the CHOP AARE in response to amino acid starvation and that ATF4 is essential for its recruitment. We also show that PCAF stimulates ATF4-driven transcription via its histone acetyltransferase domain. Thus PCAF acts as a coactivator of ATF4 and is involved in the enhancement of CHOP transcription following amino acid starvation.

The transcriptional activation of CHOP (a CCAAT/enhancer-binding protein-related gene) by amino acid deprivation involves the activating transcription factor 2 (ATF2) and the activating transcription factor 4 (ATF4) binding the amino acid response element (AARE) within the promoter. Using a chromatin immunoprecipitation approach, we report that in vivo binding of phospho-ATF2 and ATF4 to CHOP AARE are associated with acetylation of histones H4 and H2B in response to amino acid starvation. A time course analysis reveals that ATF2 phosphorylation precedes histone acetylation, ATF4 binding and the increase in CHOP mRNA. We also show that ATF4 binding and histone acetylation are two independent events that are required for the CHOP induction upon amino acid starvation. Using ATF2-deficient mouse embryonic fibroblasts, we demonstrate that ATF2 is essential in the acetylation of histone H4 and H2B in vivo. The role of ATF2 on histone H4 acetylation is dependent on its binding to the AARE and can be extended to other amino acid regulated genes. Thus, ATF2 is involved in promoting the modification of the chromatin structure to enhance the transcription of a number of amino acid-regulated genes.

The most noticeable morphological feature of the cerebellum is its folded appearance, whereby fissures separate its anterior-posterior extent into lobules. Each lobule is molecularly coded along the medial-lateral axis by parasagittal stripes of gene expression in one cell type, the Purkinje cells (PCs). Additionally, within each lobule distinct combinations of afferents terminate and supply the cerebellum with synchronized sensory and motor information. Strikingly, afferent terminal fields are organized into parasagittal domains, and this pattern bears a close relationship to PC molecular coding. Thus, cerebellum three-dimensional complexity obeys a basic coordinate system that can be broken down into morphology and molecular coding. In this review, we summarize the sequential stages of cerebellum development that produce its laminar structure, foliation, and molecular organization. We also introduce genes that regulate morphology and molecular coding, and discuss the establishment of topographical circuits within the context of the two coordinate systems. Finally, we discuss how abnormal cerebellar organization may result in neurological disorders like autism

Maternal age affects oocyte quality and early embryo development. Aberrant meiosis of oocytes and compromised early embryo development from older females could originate from defects in the nucleus, the cytoplasm, or both. Nuclear transfer has been used for decades as a tool to study nuclear-cytoplasmic interactions in early embryos, and has uncovered genomic imprinting, nuclear reprogramming, and produced animal clones. Here, we describe the technique for investigating nuclear-cyoplasmic interactions in oocytes and zygotes in female reproductive aging. Nuclear transfer can be performed efficiently and effects of the technique itself on meiosis and early embryo development are minimal as long as care is taken to minimize insult to oocytes or embryos. This protocol first focuses on use of nuclear transfer to study nucleus versus cytoplasmic origin in agingassociated meiosis defects in oocytes at the germinal vesicle (GV) stage. Then, nuclear transfer is used at the zygote stage to study nuclear and cytoplasmic abnormality and apoptosis in early development

Na+,K+-ATPase and its specific inhibitor ouabain entered the 21st century with an entirely new set of properties, that are the focuses of the present review. (i) The adhesive property of the beta-subunit explains why is Na+,K+-ATPase expressed polarizedly on one side of epithelial cells, a crucial property to explain the exchange of substances between higher organisms and the environment; (ii) Ouabain was recently recognized to be a hormone. (iii) Na+,K+-ATPase is known to act as a receptor for hormone ouabain, (iv) binding of ouabain to the Na+,K+-ATPase modifies adhesion: at high concentrations the outcome is total detachment. (v) Ouabain-resistant cells and ouabain-sensitive ones establish a special type of cell-cell interaction, so that sensitive cells withstand the presence of otherwise lethal levels of ouabain. (vi) Hormone ouabain provokes relocalization of specific molecules from the submembrane scaffold to the nucleus, where these bind to promoters of genes involved in proliferation, differentiation, migration, etc. (vii) Finally, ouabain causes a retrieval of Na+,K+-ATPase from the plasma membrane. We speculate that this would reduce the driving force that operates co- and counter-transporters, which are responsible for the exchange of substances across epithelia.