Biomedical subjects

Shaun M Cowley

Publications and source records attributed to Shaun M Cowley.

5 recordsLinked to original sources

PubMed2005

The mSin3A chromatin-modifying complex is essential for embryogenesis and T-cell development.

The corepressor mSin3A is the core component of a chromatin-modifying complex that is recruited by multiple gene-specific transcriptional repressors. In order to understand the role of mSin3A during development, we generated constitutive germ line as well as conditional msin3A deletions. msin3A deletion in the developing mouse embryo results in lethality at the postimplantation stage, demonstrating that it is an essential gene. Blastocysts derived from preimplantation msin3A null embryos and mouse embryo fibroblasts (MEFs) lacking msin3A display a significant reduction in cell division. msin3A null MEFs also show mislocalization of the heterochromatin protein, HP1alpha, without alterations in global histone acetylation. Heterozygous msin3A(+/-) mice with a systemic twofold decrease in mSin3A protein develop splenomegaly as well as kidney disease indicative of a disruption of lymphocyte homeostasis. Conditional deletion of msin3A from developing T cells results in reduced thymic cellularity and a fivefold decrease in the number of cytotoxic (CD8) T cells, while helper (CD4) T cells are unaffected. We show that CD8 development is dependent on mSin3A at a step downstream of T-cell receptor signaling and that loss of mSin3A specifically decreases survival of double-positive and CD8 T cells. Thus, msin3A is a pleiotropic gene which, in addition to its role in cell cycle progression, is required for the development and homeostasis of cells in the lymphoid lineage.

Animals↗

PubMed2004

HBP1 and Mad1 repressors bind the Sin3 corepressor PAH2 domain with opposite helical orientations.

Recruitment of the histone deacetylase (HDAC)-associated Sin3 corepressor is an obligatory step in many eukaryotic gene silencing pathways. Here we show that HBP1, a cell cycle inhibitor and regulator of differentiation, represses transcription in a HDAC/Sin3-dependent manner by targeting the mammalian Sin3A (mSin3A) PAH2 domain. HBP1 is unrelated to the Mad1 repressor for which high-resolution structures in complex with PAH2 have been described. We show that like Mad1, the HBP1 transrepression domain binds through a helical structure to the hydrophobic cleft of mSin3A PAH2. Notably, the HBP1 helix binds PAH2 in a reversed orientation relative to Mad1 and, equally unexpectedly, this is correlated with a chain reversal of the minimal Sin3 interaction motifs. These results not only provide insights into how multiple, unrelated transcription factors recruit the same coregulator, but also have implications for how sequence similarity searches are conducted.

Amino Acid Motifs↗

PubMed2004

Functional analysis of the Mad1-mSin3A repressor-corepressor interaction reveals determinants of specificity, affinity, and transcriptional response.

The recruitment of corepressors by DNA-bound repressors is likely to be a critical rate-limiting step in the transcriptional regulation of many genes. An excellent paradigm for such an interaction is the association of the basic helix-loop-helix zipper protein Mad1 with the corepressor mSin3A. When bound together, the Sin3 interaction domain (SID) of Mad1 forms extensive hydrophobic contacts with the four-helix bundle formed by the paired amphipathic helix 2 (PAH2) domain of mSin3A. Using the costructure to predict the principle residues required for binding, we have carried out an extensive mutational analysis to examine the Mad1 SID-mSin3A PAH2 interaction in vitro and in vivo. Bulky hydrophobic residues in the alpha1 (I308 and V311) and alpha2 (L329 and L332) helices of the PAH2 domain are necessary to accommodate the precise arrangement of bulky (L12) and short (A15 and A16) hydrophobic residues in the amphipathic Mad1 SID. We have also used phage display to derive an optimal SID, which shows an essentially identical arrangement of key residues. By manipulating these key residues, we have generated altered-specificity Mad1 SID mutants that bind only to a PAH2 domain with a reciprocal mutation, permitting us to demonstrate for the first time that these domains interact directly in vivo. We have also found that the integrity of the PAH1 domain affects the Mad1 SID-PAH2 interaction. It is conceivable that cross talk between different PAH domains and their binding partners helps to determine the subunit composition and order of assembly of mSin3A complexes.

Amino Acid Sequence↗

PubMed2003

Genomic binding by the Drosophila Myc, Max, Mad/Mnt transcription factor network.

The Myc/Max/Mad transcription factor network is critically involved in cell behavior; however, there is relatively little information on its genomic binding sites. We have employed the DamID method to carry out global genomic mapping of the Drosophila Myc, Max, and Mad/Mnt proteins. Each protein was tethered to Escherichia coli DNA adenine-methyltransferase (Dam) permitting methylation proximal to in vivo binding sites in Kc cells. Microarray analyses of methylated DNA fragments reveals binding to multiple loci on all major Drosophila chromosomes. This approach also reveals dynamic interactions among network members as we find that increased levels of dMax influence the extent of dMyc, but not dMnt, binding. Computer analysis using the REDUCE algorithm demonstrates that binding regions correlate with the presence of E-boxes, CG repeats, and other sequence motifs. The surprisingly large number of directly bound loci ( approximately 15% of coding regions) suggests that the network interacts widely with the genome. Furthermore, we employ microarray expression analysis to demonstrate that hundreds of DamID-binding loci correspond to genes whose expression is directly regulated by dMyc in larvae. These results suggest that a fundamental aspect of Max network function involves widespread binding and regulation of gene expression.

Animals↗

PubMed2002

Histone deacetylase 6 binds polyubiquitin through its zinc finger (PAZ domain) and copurifies with deubiquitinating enzymes.

Histone deacetylases (HDACs) are thought to function as critical mediators of transcriptional repression. However, the physiological targets and posttranslational modifications of the class II HDACs are largely unknown. Here we show that the C terminus of HDAC 6 is both necessary and sufficient for specific association with polyubiquitin. This region contains a putative zinc finger but lacks significant similarity to other known ubiquitin binding domains. Thus, we have designated this region as a PAZ domain, for Polyubiquitin Associated Zinc finger. Although the PAZ domain possesses homology with the zinc finger of deubiquitinating enzymes, it is dispensable for the deubiquitinating activity we find associated with HDAC6 following immunopurification. We also show that both HDAC 5 and HDAC 6 are ubiquitinated in vitro and in vivo. However, both of these proteins are stable in vivo and do not appear to be targeted for rapid degradation by the proteasome. Thus, HDAC6 is linked to the ubiquitin system via ubiquitin conjugation, polyubiquitin binding, and copurification with deubiquitinating enzymes.

Amino Acid Sequence↗