Daniel M. Cimbora

The Protein Network of HIV Budding

HIV release requires TSG101, a cellular factor that sorts proteins into vesicles that bud into multivesicular bodies (MVB). To test whether other proteins involved in MVB biogenesis (the class E proteins) also participate in HIV release, we identified 22 candidate human class E proteins. These proteins were connected into a coherent network by 43 different protein-protein interactions, with AIP1 playing a key role in linking complexes that act early (TSG101/ESCRT-I) and late (CHMP4/ESCRT-III) in the pathway. AIP1 also binds the HIV-1 p6(Gag) and EIAV p9(Gag) proteins, indicating that it can function directly in virus budding. Human class E proteins were found in HIV-1 particles, and dominant-negative mutants of late-acting human class E proteins arrested HIV-1 budding through plasmal and endosomal membranes. These studies define a protein network required for human MVB biogenesis and indicate that the entire network participates in the release of HIV and probably many other viruses.

The β-Globin LCR Is Not Necessary for an Open Chromatin Structure or Developmentally Regulated Transcription of the Native Mouse β-Globin Locus

The murine beta-globin locus control region (LCR) was deleted from its native chromosomal location. The approximately 25 kb deletion eliminates all sequences and structures homologous to those defined as the human LCR. In differentiated ES cells and erythroleukemia cells containing the LCR-deleted chromosome, DNasel sensitivity of the beta-globin domain is established and maintained, developmental regulation of the locus is intact, and beta-like globin RNA levels are reduced 5%-25% of normal. Thus, in the native murine beta-globin locus, the LCR is necessary for normal levels of transcription, but other elements are sufficient to establish the open chromatin structure, transcription, and developmental specificity of the locus. These findings suggest a contributory rather than dominant function for the LCR in its native location.

Genomic Targeting of Methylated DNA: Influence of Methylation on Transcription, Replication, Chromatin Structure, and Histone Acetylation

ABSTRACT We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure.

The Locus Control Region Is Necessary for Gene Expression in the Human β-Globin Locus but Not the Maintenance of an Open Chromatin Structure in Erythroid Cells

ABSTRACT Studies in many systems have led to the model that the human β-globin locus control region (LCR) regulates the transcription, chromatin structure, and replication properties of the β-globin locus. However the precise mechanisms of this regulation are unknown. We have developed strategies to use homologous recombination in a tissue culture system to examine how the LCR regulates the locus in its natural chromosomal environment. Our results show that when the functional components of the LCR, as defined by transfection and transgenic studies, are deleted from the endogenous β-globin locus in an erythroid background, transcription of all β-globin genes is abolished in every cell. However, formation of the remaining hypersensitive site(s) of the LCR and the presence of a DNase I-sensitive structure of the β-globin locus are not affected by the deletion. In contrast, deletion of 5′HS5 of the LCR, which has been suggested to serve as an insulator, has only a minor effect on β-globin transcription and does not influence the chromatin structure of the locus. These results show that the LCR as currently defined is not necessary to keep the locus in an “open” conformation in erythroid cells and that even in an erythroid environment an open locus is not sufficient to permit transcription of the β-like globin genes.

Long-Distance Control of Origin Choice and Replication Timing in the Human β-Globin Locus Are Independent of the Locus Control Region

ABSTRACT DNA replication in the human β-globin locus is subject to long-distance regulation. In murine and human erythroid cells, the human locus replicates in early S phase from a bidirectional origin located near the β-globin gene. This Hispanic thalassemia deletion removes regulatory sequences located over 52 kb from the origin, resulting in replication of the locus from a different origin, a shift in replication timing to late S phase, adoption of a closed chromatin conformation, and silencing of globin gene expression in murine erythroid cells. The sequences deleted include nuclease-hypersensitive sites 2 to 5 (5′HS2-5) of the locus control region (LCR) plus an additional 27-kb upstream region. We tested a targeted deletion of 5′HS2-5 in the normal chromosomal context of the human β-globin locus to determine the role of these elements in replication origin choice and replication timing. We demonstrate that the 5′HS2-5-deleted locus initiates replication at the appropriate origin and with normal timing in murine erythroid cells, and therefore we conclude that 5′HS2-5 in the classically defined LCR do not control replication in the human β-globin locus. Recent studies also show that targeted deletion of 5′HS2-5 results in a locus that lacks globin gene expression yet retains an open chromatin conformation. Thus, the replication timing of the locus is closely correlated with nuclease sensitivity but not globin gene expression.

Characterization of the Cellular and Antitumor Effects of MPI-0479605, a Small-Molecule Inhibitor of the Mitotic Kinase Mps1

Mps1 is a dual specificity protein kinase that is essential for the bipolar attachment of chromosomes to the mitotic spindle and for maintaining the spindle assembly checkpoint until all chromosomes are properly attached. Mps1 is expressed at high levels during mitosis and is abundantly expressed in cancer cells. Disruption of Mps1 function induces aneuploidy and cell death. We report the identification of MPI-0479605, a potent and selective ATP competitive inhibitor of Mps1. Cells treated with MPI-0479605 undergo aberrant mitosis, resulting in aneuploidy and formation of micronuclei. In cells with wild-type p53, this promotes the induction of a postmitotic checkpoint characterized by the ATM- and RAD3-related-dependent activation of the p53–p21 pathway. In both wild-type and p53 mutant cells lines, there is a growth arrest and inhibition of DNA synthesis. Subsequently, cells undergo mitotic catastrophe and/or an apoptotic response. In xenograft models, MPI-0479605 inhibits tumor growth, suggesting that drugs targeting Mps1 may have utility as novel cancer therapeutics. Mol Cancer Ther; 10(12); 2267–75. ©2011 AACR.

The Control of Mammalian DNA Replication: A Brief History of Space and Timing

The existence of G1 steps at which origin choice and replication timing are programmed in mammalian nuclei was demonstrated using a heterologous system based on Xenopus extracts. In this system, the origin decision point correlates with ORC assembly on chromatin, and the temporal decision point coincides with the postmitotic repositioning of chromosomal domains in the nucleus. It is likely that these control steps identified in vitro reflect regulatory events in living cells. However, although we do not yet have a complete picture, potential differences among metazoan replication control mechanisms are already apparent. For example, the number and spatial arrangement of replication foci differs between mammalian primary cells and cell lines (Kennedy et al., 2000xKennedy, B.K., Barbie, D.A., Classon, M., Dyson, N., and Harlow, E. Genes Dev. 2000; 14: 2855–2868Crossref | PubMed | Scopus (212)See all References(Kennedy et al., 2000), and different profiles of Orc protein expression during the cell cycle are evident among metazoans (Natale et al., 2000xNatale, D.A., Li, C.-J., Sun, W.-H., and DePamphilis, M.L. EMBO J. 2000; 19: 2728–2738Crossref | PubMedSee all ReferencesNatale et al., 2000 and references therein), suggesting different replication control mechanisms may exist. These differences emphasize the need for caution when extrapolating results from one organism or experimental system to another. The identification of further similarities and the reconciliation of differences among these diverse systems will be important for refining models of replication control in higher eukaryotes.

2-Anilino-4-aryl-1,3-thiazole inhibitors of valosin-containing protein (VCP or p97).

Valosin-containing protein (VCP; also known as p97) is a member of the AAA ATPase family with a central role in the ubiquitin-degradation of misfolded proteins. VCP also exhibits antiapoptotic function and metastasis via activation of nuclear factor kappa-B signaling pathway. We have discovered that 2-anilino-4-aryl-1,3-thiazoles are potent drug-like inhibitors of this enzyme. The identified compounds show low nanomolar VCP potency, demonstrate SAR trends, and show activity in a mechanism based cellular assay. This series of compounds represents the first steps towards a novel, small molecule VCP inhibitor as a cancer therapeutic.

Discovery of (2S)-1-[4-(2-{6-amino-8-[(6-bromo-1,3-benzodioxol-5-yl)sulfanyl]-9H-purin-9-yl}ethyl)piperidin-1-yl]-2-hydroxypropan-1-one (MPC-3100), a purine-based Hsp90 inhibitor.

Modulation of Hsp90 (heat shock protein 90) function has been recognized as an attractive approach for cancer treatment, since many cancer cells depend on Hsp90 to maintain cellular homeostasis. This has spurred the search for small-molecule Hsp90 inhibitors. Here we describe our lead optimization studies centered on the purine-based Hsp90 inhibitor 28a containing a piperidine moiety at the purine N9 position. In this study, key SAR was established for the piperidine N-substituent and for the congeners of the 1,3-benzodioxole at C8. These efforts led to the identification of orally bioavailable 28g that exhibits good in vitro profiles and a characteristic molecular biomarker signature of Hsp90 inhibition both in vitro and in vivo. Favorable pharmacokinetic properties along with significant antitumor effects in multiple human cancer xenograft models led to the selection of 28g (MPC-3100) as a clinical candidate.

Oxindole derivatives as inhibitors of TAK1 kinase

Several series of oxindole analogues were synthesized and screened for inhibitory activity against transforming growth factor-β-activating kinase 1 (TAK1). Modifications around several regions of the lead molecules were made, with a distal hydroxyl group in the D region being critical for activity. The most potent compound 10 shows an IC(50) of 8.9 nM against TAK1 in a biochemical enzyme assay, with compounds 3 and 6 showing low micromolar cellular inhibition.