Faculty Bibliography

Cyclin-dependent kinase 5 (Cdk5), a serine/threonine kinase that displays kinase activity predominantly in neurons, is activated by two non-cyclin activators, p35 or p39. Here, we report a physical and functional interaction between the Cdk5/p35 complex and mouse Sds3 (mSds3), an essential component of mSin3-histone deacetylase (HDAC) co-repressor complex. mSds3 binds to p35 both in vitro and in vivo, enabling active Cdk5 to phosphorylate mSds3 at serine 228. A mSds3 S228A mutant retained mSin3 binding activity, but its dimerization was not greatly enhanced by p35 when compared with wild type. Notably, p35 overexpression augmented mSds3-mediated transcriptional repression in vitro. Interestingly, mutational studies revealed that the ability of exogenous mSds3 to rescue cell growth and viability in mSds3 null cells correlates with its ability to be phosphorylated by Cdk5. The identification of mSds3 as a substrate of the Cdk5/p35 complex reveals a new regulatory mechanism in controlling the mSin3-HDAC transcriptional repressor activity and provides a new potential therapeutic means to inhibit specific HDAC activities in disease

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is critical for EBV immortalization of infected B lymphocytes and can coactivate the EBV LMP1 promoter with EBNA2. EBNA3C amino acids 365 to 545 are necessary and sufficient for coactivation and are required for SUMO-1 and SUMO-3 interaction. We found that EBNA3C but not EBNA3CDelta343-545 colocalized with SUMO-1 in nuclear bodies and was modified by SUMO-2, SUMO-3, and SUMO-1. EBNA3C amino acids 545 to 628 and amino acids 30 to 365 were also required for EBNA3C sumolation and nuclear body localization but were dispensable for coactivation, indicating that EBNA3C sumolation is not required for coactivation. Furthermore, EBNA3C amino acids 476 to 992 potently coactivated with EBNA2 but EBNA3C amino acids 516 to 922 lacked activity, indicating that amino acids 476 to 515 are critical for coactivation. EBNA3C amino acids 476 to 515 include DDDVIEV(507-513), which are similar to SUMO-1 EEDVIEV(84-90). EBNA3C m1 and m2 point mutations, DDD(507-509) mutated to AAA and DVIEVID(509-513) mutated to AVIAVIA, respectively, diminished SUMO-1 and SUMO-3 interaction in directed yeast two-hybrid and glutathione S-transferase pulldown assays. Furthermore, EBNA3C m1 and m2 did not coactivate the LMP1 promoter with EBNA2. Overexpression of wild-type SUMO-1, SUMO-3, and the SUMO-conjugating enzyme UBC9 coactivated the LMP1 promoter with EBNA2. Since EBNA2 activation is dependent on p300/CBP, the possible effect of EBNA3C on p300-mediated transcription was assayed. EBNA3C potentiated transcription of p300 fused to a heterologous DNA binding domain, whereas EBNA3C m1 and m2 did not. All of these data are consistent with a model in which EBNA3C upregulates EBNA2-mediated gene activation by binding to a sumolated repressor and inhibiting repressive effects on p300/CBP and other transcription factor(s) at EBNA2-regulated promoters

The histone code guides many aspects of chromosome biology including the equal distribution of chromosomes during cell division. In the chromatin domains surrounding the centromere, known as pericentric heterochromatin, histone modifications, particularly deacetylation and methylation, appear to be essential for proper chromosome segregation. However, the specific factors and their precise roles in this highly orchestrated process remain under active investigation. Here, we report that germ-line or somatic deletion of mSds3, an essential component of the functional mSin3/HDAC corepressor complex, generates a cell-lethal condition associated with rampant aneuploidy, defective karyokinesis, and consequently, a failure of cytokinesis. mSds3-deficient cells fail to deacetylate and methylate pericentric heterochromatin histones and to recruit essential heterochromatin-associated proteins, resulting in aberrant associations among heterologous chromosomes via centromeric regions and consequent failure to properly segregate chromosomes. Mutant mSds3 molecules that are defective in mSin3 binding fail to rescue the mSds3 null phenotypes. On the basis of these findings, we propose that mSds3 and its associated mSin3/HDAC components play a central role in initiating the cascade of pericentric heterochromatin-specific modifications necessary for the proper distribution of chromosomes during cell division in mammalian cells

Histone deacetylation plays a central role in the regulation of genes linked to virtually all biological processes. This modification reaction is dependent on a family of related histone deacetylases (HDACs), which function as key components of large multiprotein complexes involved in the development of normal and neoplastic cells. The mechanisms regulating HDACs and their roles in such processes are not understood, and these form the major focus for the current study. Here, in the course of assessing possible post-translational modifications of HDAC1, we demonstrated that HDAC1 is a substrate for SUMO-1 (small ubiquitin-related modifier) modification in vitro and in vivo. The HDAC1 lysines targeted for modification were identified as C-terminal Lys-444 and Lys-476, which are also present in mammalian HDAC2 and lower vertebrate HDAC1/2 orthologs yet absent from other HDAC family members, pointing to a means of differential regulation among HDAC proteins. Mutation of these target residues (lysine to arginine substitution) profoundly reduced HDAC1-mediated transcriptional repression in reporter assays without affecting HDAC1 ability to associate with mSin3A and eliminated HDAC1-induced cell cycle and apoptotic responses upon overexpression. Together, the results demonstrate that HDAC1 is modified by SUMO-1, and this modification can dramatically affect HDAC1 activity in a number of surrogate biological assays

Silencing of gene transcription involves local chromatin modification achieved through the local recruitment of large multiprotein complexes containing histone deacetylase (HDAC) activity. The mammalian corepressors mSin3A and mSin3B have been shown to play a key role in this process by tethering HDACs 1 and 2 to promoter-bound transcription factors. Similar mechanisms appear to be operative in yeast, in which epistasis experiments have established that the mSin3 and HDAC orthologs (SIN3 and RPD3), along with a novel protein, SDS3, function in the same repressor pathway. Here, we report the identification of a component of the mSin3-HDAC complex that bears homology to yeast SDS3, physically associates with mSin3 proteins in vivo, represses transcription in a manner that is partially dependent on HDAC activity, and enables HDAC1 catalytic activity in vivo. That key physical and functional properties are also shared by yeast SDS3 underscores the central role of the Sin3-HDAC-Sds3 complex in eukaryotic cell biology, and the discovery of mSds3 in mammalian cells provides a new avenue for modulating the activity of this complex in human disease

The 2'-5' oligoadenylate synthetases (OAS) represent a family of interferon (IFN)-induced proteins implicated in the antiviral action of IFN. When activated by double-stranded (ds) RNA, these proteins polymerize ATP into 2'-5' linked oligomers with the general formula pppA(2'p5'A)n, n greater than or = 1. Three forms of human OAS have been described corresponding to proteins of 40/46, 69/71, and 100 kDa. These isoforms are encoded by three distinct genes clustered on chromosome 12 and exhibit differential constitutive and IFN-inducible expression. Here we describe the structural and functional analysis of the gene encoding the large form of human OAS. This gene has 16 exons with exon/intron boundaries that are conserved among the different isoforms of the human OAS family, reflecting the evolutionary link among them. The promoter region of the p100 gene is composed of multiple features conferring direct inducibility not only by IFNs but also by TNF and all-trans retinoic acid. In contrast, the induction of the p100 promoter by dsRNA is indirect and requires IFN type I production.

The c-Myc oncoprotein plays an important role in the growth and proliferation of normal and neoplastic cells. To execute these actions, c-Myc is thought to regulate functionally diverse sets of genes that directly govern cellular mass and progression through critical cell cycle transitions. Here, we provide several lines of evidence that c-Myc promotes ubiquitin-dependent proteolysis by directly activating expression of the Cul1 gene, encoding a critical component of the ubiquitin ligase SCF(SKP2). The cell cycle inhibitor p27(kip1) is a known target of the SCF(SKP2) complex, and Myc-induced Cul1 expression matched well with the kinetics of declining p27(kip1) protein. Enforced Cul1 expression or antisense neutralization of p27(kip1) was capable of overcoming the slow-growth phenotype of c-Myc null primary mouse embryonic fibroblasts (MEFs). In reconstitution assays, the addition of in vitro translated Cul1 protein alone was able to restore p27(kip1) ubiquitination and degradation in lysates derived from c-myc(-/-) MEFs or density-arrested human fibroblasts. These functional and biochemical data provide a direct link between c-Myc transcriptional regulation and ubiquitin-mediated proteolysis and together support the view that c-Myc promotes G(1) exit in part via Cul1-dependent ubiquitination and degradation of the CDK inhibitor, p27(kip1)

Myc and Mad family proteins regulate multiple biological processes through their capacity to influence gene expression directly. Here we show that the basic regions of Myc and Mad proteins are not functionally equivalent in oncogenesis, have separable E-box-binding activities and engage both common and distinct gene targets. Our data support the view that the opposing biological actions of Myc and Mxi1 extend beyond reciprocal regulation of common gene targets. Identification of differentially regulated gene targets provides a framework for understanding the mechanism through which the Myc superfamily governs the growth, proliferation and survival of normal and neoplastic cells

The PLZF gene was identified first by its fusion with the retinoic acid receptor alpha gene in the t(11;17) translocation associated with a retinoic acid resistant form of acute promyelocytic leukemia (APL). It encodes a kruppel-like zinc finger protein with a POZ domain shared with a subset of regulatory proteins including the BCL6 leukemogenic protein. PLZF, like BCL6, strongly represses transcription initiated from different promoters. Here we show that PLZF associates in vitro and in vivo with the Mad co-repressor mSin3A and the histone deacetylase HDAC1. Two domains in PLZF and the PAH1 structure of mSin3A mediate these interactions. Trichostatin A, a specific inhibitor of histone deacetylases, significantly reduces PLZF repression. These data strongly suggest that, like nuclear receptors and Mad, PLZF represses transcription by recruiting a histone deacetylase through the SMRT-mSin3-HDAC co-repressor complex. We also show that BCL6 associates with HDAC1 indicating that this type of regulation might be common to POZ/Zinc finger proteins involved in human leukemias. This work supports a role for deregulated histone deacetylation in the development of both lymphoid and myeloid neoplasia in human and suggests that targeted histone deacetylase inhibitors may be useful for treatment of certain types of malignancies