Lisa Ann Cirillo

Opening of Compacted Chromatin by Early Developmental Transcription Factors HNF3 (FoxA) and GATA-4

The transcription factors HNF3 (FoxA) and GATA-4 are the earliest known to bind the albumin gene enhancer in liver precursor cells in embryos. To understand how they access sites in silent chromatin, we assembled nucleosome arrays containing albumin enhancer sequences and compacted them with linker histone. HNF3 and GATA-4, but not NF-1, C/EBP, and GAL4-AH, bound their sites in compacted chromatin and opened the local nucleosomal domain in the absence of ATP-dependent enzymes. The ability of HNF3 to open chromatin is mediated by a high affinity DNA binding site and by the C-terminal domain of the protein, which binds histones H3 and H4. Thus, factors that potentiate transcription in development are inherently capable of initiating chromatin opening events.

Binding of the winged-helix transcription factor HNF3 to a linker histone site on the nucleosome.

The transcription factor HNF3 and linker histones H1 and H5 possess winged‐helix DNA‐binding domains, yet HNF3 and other fork head‐related proteins activate genes during development whereas linker histones compact DNA in chromatin and repress gene expression. We compared how the two classes of factors interact with chromatin templates and found that HNF3 binds DNA at the side of nucleosome cores, similarly to what has been reported for linker histone. A nucleosome structural binding site for HNF3 is occupied at the albumin transcriptional enhancer in active and potentially active chromatin, but not in inactive chromatin in vivo. While wild‐type HNF3 protein does not compact DNA extending from the nucleosome, as does linker histone, site‐directed mutants of HNF3 can compact nucleosomal DNA if they contain basic amino acids at positions previously shown to be essential for nucleosomal DNA compaction by linker histones. The results illustrate how transcription factors can possess special nucleosome‐binding activities that are not predicted from studies of factor interactions with free DNA.

An early developmental transcription factor complex that is more stable on nucleosome core particles than on free DNA.

In vivo footprinting studies have shown that transcription factor binding sites for HNF3 and GATA-4 are occupied on the albumin gene enhancer in embryonic endoderm, prior to the developmental activation of liver gene transcription. We have investigated how these factors can stably occupy silent chromatin. Remarkably, we find that HNF3, but not GATA-4 or a GAL4 control protein, binds far more stably to nucleosome core particles than to free DNA. In the presence of HNF3, GATA-4 binds stably to an HNF3-positioned nucleosome. Histone acetylation does not affect HNF3 binding. This is evidence for stable nucleosome binding by a transcription factor and shows that a winged helix protein is sufficient to initiate the assembly of an enhancer complex on nonacetylated nucleosomes.

Chromatin Opening and Stable Perturbation of Core Histone:DNA Contacts by FoxO1

FoxO1, a member of the forkhead rabdomyosarcoma (FoxO) subfamily of transcription factors, binds DNA via a highly conserved winged-helix “forkhead box” motif used by other regulatory proteins to mediate their effects through chromatin binding and remodeling. To examine how FoxO1 regulates target genes in chromatin, we studied the binding of purified recombinant FoxO1 protein to nucleosome particles and chromatin arrays containing the insulin-like growth factor-binding protein 1 promoter. We found that FoxO1 is able to bind to its cognate sites within the insulin-like growth factor-binding protein 1 promoter on a nucleosome. This binding stably perturbs core histone:DNA contacts extending up- and downstream from sites of FoxO1 binding without disrupting the underlying core particle. FoxO1 is able to harness these capabilities to bind to and de-condense linker histone-compacted chromatin arrays. Chromatin opening by FoxO1 requires both the N and C termini of the protein, which are also required for high affinity core histone binding and, in the case of the N terminus, nucleosome perturbation. We suggest that the chromatin binding and remodeling functions revealed here for FoxO1 endow all FoxO factors with the ability to initiate and dynamically modulate active chromatin states, enabling their diverse roles as gene regulatory factors in metabolism, cell survival, apoptosis, cell cycle progression, DNA repair, and protection against oxidative stress.

Integrase-Specific Enhancement and Suppression of Retroviral DNA Integration by Compacted Chromatin Structure In Vitro

ABSTRACT Integration of viral DNA into the host chromosome is an obligatory step in retroviral replication and is dependent on the activity of the viral enzyme integrase. To examine the influence of chromatin structure on retroviral DNA integration in vitro, we used a model target comprising a 13-nucleosome extended array that includes binding sites for specific transcription factors and can be compacted into a higher-ordered structure. We found that the efficiency of in vitro integration catalyzed by human immunodeficiency virus type 1 (HIV-1) integrase was decreased after compaction of this target with histone H1. In contrast, integration by avian sarcoma virus (ASV) integrase was more efficient after compaction by either histone H1 or a high salt concentration, suggesting that the compacted structure enhances this reaction. Furthermore, although site-specific binding of transcription factors HNF3 and GATA4 blocked ASV DNA integration in extended nucleosome arrays, local opening of H1-compacted chromatin by HNF3 had no detectable effect on integration, underscoring the preference of ASV for compacted chromatin. Our results indicate that chromatin structure affects integration site selection of the HIV-1 and ASV integrases in opposite ways. These distinct properties of integrases may also affect target site selection in vivo, resulting in an important bias against or in favor of integration into actively transcribed host DNA.

The CBP Bromodomain and Nucleosome Targeting Are Required for Zta-Directed Nucleosome Acetylation and Transcription Activation

ABSTRACT The Epstein-Barr virus (EBV)-encoded lytic activator Zta is a bZIP protein that can stimulate nucleosomal histone acetyltransferase (HAT) activity of the CREB binding protein (CBP) in vitro. We now show that deletion of the CBP bromo- and C/H3 domains eliminates stimulation of nucleosomal HAT activity in vitro and transcriptional coactivation by Zta in transfected cells. In contrast, acetylation of free histones was not affected by the addition of Zta or by deletions in the bromo or C/H3 domain of CBP. Zta stimulated acetylation of oligonucleosomes assembled on supercoiled DNA and dinucleosomes assembled on linear DNA, but Zta-stimulated acetylation was significantly reduced for mononucleosomes. Western blotting and amino-terminal protein sequencing indicated that all lysine residues in the H3 and H4 amino-terminal tails were acetylated by CBP and enhanced by the addition of Zta. Histone acetylation was also dependent upon the Zta basic DNA binding domain, which could not be substituted with the homologous basic region of c-Fos, indicating specificity in the bZIP domain nucleosome binding function. Finally, we show that Zta and CBP colocalize to viral immediate-early promoters in vivo and that overexpression of Zta leads to a robust increase in H3 and H4 acetylation at various regions of the EBV genome in vivo. Furthermore, deletion of the CBP bromodomain reduced stable CBP-Zta complex formation and histone acetylation at Zta-responsive viral promoters in vivo. These results suggest that activator- and bromodomain-dependent targeting to oligonucleosomal chromatin is required for stable promoter-bound complex formation and transcription activity.

Sequence-Specific Capture of Protein-DNA Complexes for Mass Spectrometric Protein Identification

The regulation of gene transcription is fundamental to the existence of complex multicellular organisms such as humans. Although it is widely recognized that much of gene regulation is controlled by gene-specific protein-DNA interactions, there presently exists little in the way of tools to identify proteins that interact with the genome at locations of interest. We have developed a novel strategy to address this problem, which we refer to as GENECAPP, for Global ExoNuclease-based Enrichment of Chromatin-Associated Proteins for Proteomics. In this approach, formaldehyde cross-linking is employed to covalently link DNA to its associated proteins; subsequent fragmentation of the DNA, followed by exonuclease digestion, produces a single-stranded region of the DNA that enables sequence-specific hybridization capture of the protein-DNA complex on a solid support. Mass spectrometric (MS) analysis of the captured proteins is then used for their identification and/or quantification. We show here the development and optimization of GENECAPP for an in vitro model system, comprised of the murine insulin-like growth factor-binding protein 1 (IGFBP1) promoter region and FoxO1, a member of the forkhead rhabdomyosarcoma (FoxO) subfamily of transcription factors, which binds specifically to the IGFBP1 promoter. This novel strategy provides a powerful tool for studies of protein-DNA and protein-protein interactions.

Stable Chromatin Binding Prevents FoxA Acetylation, Preserving FoxA Chromatin Remodeling

FoxA1–3 (formerly HNF3α, -β, and -γ), members of the FoxA subfamily of forkhead transcription factors, function as initial chromatin-binding and chromatin-remodeling factors in a variety of tissues, including liver and pancreas. Despite essential roles in development and metabolism, regulation of FoxA factors is not well understood. This study examines a potential role for acetylation in the regulation of FoxA chromatin binding and remodeling. Using in silico analysis, we have identified 11 putative p300 acetylation sites within FoxA1, five of which are located within wings 1 and 2 of its winged-helix DNA-binding domain. These polypeptide structures stabilize FoxA DNA and chromatin binding, and we have demonstrated that acetylation attenuates FoxA binding to DNA and diminishes its ability to remodel chromatin. FoxA acetylation is inhibited by chromatin binding. We propose a model whereby stable chromatin binding protects the FoxA DNA-binding domain from acetylation to preserve chromatin binding and remodeling by FoxA factors in the absence of extracellular cues.

Dynamic association of gammaherpesvirus DNA with core histone during de novo lytic infection of primary cells

Association of herpesvirus DNA with histones has important implications for lytic and latent infections; thus herpesviruses arbitrate interactions with histones to productively infect host cells. While regulation of alpha and betaherpesvirus chromatin during lytic infection has been actively investigated, very little is known about interaction of gammaherpesvirus DNA with histones upon de novo lytic infection. Murine gammaherpesvirus-68 (MHV68) is a rodent pathogen that offers a tractable system to study gammaherpesvirus lytic infection in primary cells. In this study we report that MHV68 promoter and orilyt sequences underwent dynamic association with histone H3 during de novo lytic infection of primary macrophages and fibroblasts. Similar to HSV-1, the degree of MHV68 DNA association with histone H3 was dependent on the multiplicity of infection and was further regulated by viral DNA synthesis. Our work sets a precedent for future studies of gammaherpesvirus chromatin during de novo lytic infection.

Gammaherpesvirus gene expression and DNA synthesis are facilitated by viral protein kinase and histone variant H2AX.

Gammaherpesvirus protein kinases are an attractive therapeutic target as they support lytic replication and latency. Via an unknown mechanism these kinases enhance expression of select viral genes and DNA synthesis. Importantly, the kinase phenotypes have not been examined in primary cell types. Mouse gammaherpesvirus-68 (MHV68) protein kinase orf36 activates the DNA damage response (DDR) and facilitates lytic replication in primary macrophages. Significantly, H2AX, a DDR component and putative orf36 substrate, enhances MHV68 replication. Here we report that orf36 facilitated expression of RTA, an immediate early MHV68 gene, and DNA synthesis during de novo infection of primary macrophages. H2AX expression supported efficient RTA transcription and phosphorylated H2AX associated with RTA promoter. Furthermore, viral DNA synthesis was attenuated in H2AX-deficient macrophages, suggesting that the DDR system was exploited throughout the replication cycle. The interactions between a cancer-associated gammaherpesvirus and host tumor suppressor system have important implications for the pathogenesis of gammaherpesvirus infection.