My research program is centered on investigating the regulation of gene expression in eukaryotic cells at the level of transcription. My laboratory uses two model organisms/cells, yeast and human T-cells. The model promoters are PHO5 and the HTLV-1 LTR. We use both in vivo and biochemical approaches to address the mechanism of transcriptional repression and activation in chromatin.

Initially, the research in my laboratory focused on the role of chromatin structure in transcriptional regulation using the yeast system. We have developed a yeast chromatin assembly system that is the most widely used by other laboratories conducting analysis of transcriptional regulation in a chromatin context. We have employed this system to investigate the mechanism of PHO5 transcriptional regulation.

The expansion of my research program into human T-cells and HTLV-1 Tax transactivation in collaboration with Jenny Nyborg has been rapid and successful. Finally, the addition of Karolin Luger and Jeff Hansen to the Biochemistry faculty has greatly expanded the possibilities for collaborative projects in chromatin structure and function. The recent Keck Award validates the uniqueness of the environment within this department for my research program.

PHO5 Regulation

The PHO5 gene promoter has become an important model for the study of gene regulation in the context of chromatin. The PHO5 gene is repressed in high phosphate medium through the presence of four translationally positioned nucleosomes on the promoter. Upon PHO5 activation, the nucleosomes are displaced (Figure 1). This chromatin transition has been extensively studied in vivo. However, the mechanism of nucleosome displacement is still unclear.

Our first goal was to establish a yeast in vitro chromatin assembly system in order to investigate biochemically the mechanism of transcriptional regulation in a chromatin context. At that time, no in vitro yeast chromatin assembly system existed. Establishing this system required developing a yeast histone purification method, as well as developing a procedure for properly assembling them into nucleosomes on transcription templates (Pilon et al., 1997; Wongwisansri and Laybourn, 2004). We have used templates reconstituted into chromatin with purified native and recombinant yeast core histones to investigate the process of nucleosome remodeling, a prerequisite for transcription activation, on the PHO5 promoter. Footprinting analysis showed that intrinsic properties of the promoter DNA are sufficient for translational positioning of nucleosomes, which approximates that seen in vivo. We have found that both Pho4p and Pho2p can bind their cognate sites on chromatin-assembled templates without the aid of remodeling factors. However, nucleosome remodeling by these transcriptional activators requires an ATP dependent activity in a yeast nuclear extract fraction. Finally, transcriptional activation on chromatin templates requires acetyl CoA in addition to these other activities and cofactors. We have found that, under these conditions, the histones are converted from a completely unacetylated state to a fully acetylated state. These findings indicate that transcriptional activation requires Pho4p, Pho2p, nucleosome remodeling, and nucleosome acetylation. Further, DNA binding, nucleosome remodeling, and transcriptional activation are separable steps. These findings have been published in the Journal of Biological Chemistry (Terrell et al. 2002). I have recently established collaborations with the laboratories of Mike Kladde (Texas A&M Univ.) and Blaine Bartholmew (Univ. of IL, Carbondale) to obtain a more detailed understanding of the mechanism of nucleosome positioning and remodeling on the PHO5 promoter. This collaboration has already generated a manuscript submitted for publication.

Histone deacetylase Rpd3p functions as a transcriptional repressor of a diverse set of genes including PHO5. We have identified a novel role for RPD3 in the regulation of phosphate transporter Pho84p retention in the cytoplasmic membrane (Wongwisansri and Laybourn 2005). We found that under repressing conditions (+Pi), PHO5 expression is increased in a pho4Δ rpd3Δ strain demonstrating PHO regulatory pathway independence. However, the effect of RPD3 disruption on PHO5 activation kinetics is dependent on the PHO regulatory pathway. Upon switching to activating conditions (-Pi), PHO5 transcripts accumulated more rapidly in rpd3Δ cells. This more rapid response correlates with a defect in phosphate uptake due to premature recycling of Pho84p, the high affinity phosphate symporter (Figure 2). Thus, RPD3 also participates in PHO5 regulation through a previously unidentified effect on maintenance of high affinity phosphate uptake during phosphate starvation. We propose that Rpd3p has a negative role in the regulation of Pho84p endocytosis.

Tax-mediated transcriptional activation in a chromatin context

We have established a collaboration with the Nyborg and Luger laboratories to investigate the role of CBP (CREB binding protein) in Tax/CREB transactivation (Figure 3). Efficient transcription of the human T-cell leukemia virus (HTLV-I) genome requires Tax, a virally encoded oncogenic transcription factor, in complex with the cellular transcription factor CREB and the coactivators p300/CBP. CBP has been shown to be a transcriptional co-activator and to contain histone acetyltransferase (HAT) activity. In previous studies, the Tax/CREB complex was shown to recruit CBP to the HTLV-1 promoter. In addition, CBP mediated activation was shown to result in promoter localized histone acetylation. Finally, recapitulating CBP mediated transcriptional activation in vitro required the use of a chromatin-assembled template.

To examine Tax transactivation in vitro, we used a chromatin assembly system that included recombinant core histones. The addition of Tax, CREB, and p300 to the chromatin assembled HTLV-I promoter activated transcription several hundred-fold. Chromatin templates selectively lacking amino terminal histone tails demonstrated enhanced transcriptional activation by Tax and CREB, with significantly reduced dependence on p300 and acetyl CoA. Interestingly, Tax/CREB activation from the tailless chromatin templates retained a substantial requirement for acetyl CoA, indicating a role for acetyl CoA beyond histone acetylation. These data indicate that during Tax transcriptional activation, the amino terminal histone tails are key acetylation targets, but are not the only targets. In addition, our results suggest that tail deletion and acetylation are functionally equivalent (at least in vitro). These results are published in Molecular Cell Biology (Georges et al. 2002). More recently we have demonstrated that the Tax-CBP/p300 interaction is critical to HTLV-1 transcriptional activation on chromatin. In addition, we find that there is a significant level of this coactivator in our transcription nuclear extract. Intriguingly, we have determined that the primary CBP/p300 acetyltransferase target is not the core histone tails even though modification of this substrate is required for transcriptional activation on chromatin templates (Georges et al. 2003). This finding indicates a requirement for another, as yet unidentified, histone acetylatransferase in Tax transactivation.

A recent line of investigation is into the role of core histone variants in gene regulation, in collaboration with the Luger laboratory. When we assemble chromatin with H2A.Bbd, a core histone variant deficient in Barr bodies and thought to form less repressive chromatin, we obtain chromatin templates with a more relaxed structure and reduced ability to repress Tax/CREB activation (Bao et al. 2004). We are now investigating in greater detail how H2A.Bbd and other core histone variants affect chromatin structure and transcriptional regulation.

We assayed for the presence of Tax, CBP/p300, and a range of DNA-binding transcription regulatory factors and investigated the extent of histone modification on the HTLV-1 promoter in infected T-cells (Lemasson et al. 2002). Tax, CREB, CBP, and p300 are clearly associated with the promoter. In addition, CREB-2, ATF-1, ATF-2, and AP-1 are bound. The N-terminal tails of histones H3 and H4 are highly acetylated on the HTLV-1 genome. Interestingly, histone deacetylases HDAC-1 and HDAC-2 also are bound to the promoter. Consistent with their presence, treatment of infected T-cells with deacetylase inhibitors increases H4 acetylation and HTLV-1 transcript levels. These findings verify the biological significance of CBP/p300 HAT activity in Tax transcriptional activation. In addition, we have established our ability to determine the protein occupancy and histone modification state on the HTLV-1 promoter in human T-cells. More recently, we have demonstrated that both the 5' and 3' LTRs are actively transcribed and have similar, but not identical, distributions of transcriptional regulatory factors (Lemasson et al. 2004). The most striking differences are in HDAC occupancy and the data suggests that regulation is at least partly due to mutually exclusive binding by HDACs and Tax. Finally, chromatin immunoprecipitation analysis of the level of activating core histone tail modifications on the inactive and active HTLV-1 promoter unexpectedly indicates that they decrease. The answer to this apparent paradox was provided by our demonstration that nucleosomes are evicted from the promoter and transcribed region upon Tax activation (Lemasson et al., submitted). We are currently investigating the mechanism and factor requirements. Our results thus far suggest that these SWI/SNF complexes function to maintain transcriptionally silent chromatin architecture at the HTLV I promoter. Alternatively, upon activation by Tax BRG1 and hBRM initiate nucleosome eviction and then dissociate from the promoter along with the nucleosomes. These two models are not mutually exclusive and the SWI/SNF complexes may participate in both repression and activation of the HTLV-I promoter.

In the same in vivo study we determined that H1 is associated with the chromosomally integrated HTLV I LTR at a level similar to that of the β-globin gene in the absence of Tax and is depleted along with the core histone upon Tax expression. Therefore, to obtain a complete understanding of the mechanism Tax transactivation, it is imperative to study this process in the context of chromatin templates incorporating histone H1. Our laboratory has obtained funding for investigating Tax-mediated transcriptional activation of the HTLV-1 LTR promoter in H1-containing chromatin. We have succeeded in assembling HTLV-1 chromatin templates containing one H1 per nucleosome. Interestingly, we find that incorporation of H1 into the HTLV I promoter chromatin template reduces overall basal and activated transcription, but significantly increases the fold activation by Tax, relative to that observed on chromatin templates without H1. More importantly, on H1-containing chromatin, HTLV-I activation becomes highly dependent on Tax. The combined effects of Tax and p300 on HTLV-I transcriptional activation are more than additive, increasing transcription up to 900-fold. Thus, chromatin templates assembled with H1 more accurately mirror the dramatic level of Tax activation and the strict Tax dependence that is observed in living cells. Current experiments are focused on determining the mechanism for the strong Tax-dependence of chromatin templates assembled with H1 and addressing the role of CBP/p300 and histone tail acetylation in Tax transactivation.

Histone Gene Expression is Repressed in HTLV-I infected and Tax-expressing T Cell Lines

Initially, we hypothesized that HTLV-I expression modified the expression level or modification state of linker histone H1 isoforms. We were surprised to find that HTLV-I infection as well as Tax expression alone result in reduction of histone H1 and core histone protein and mRNA levels in human T-cell lines. HTLV-I Tax functions to short circuit the normal regulation of cell cycle progression by abrogating the need for mitogen stimulation and blocking checkpoint controls, resulting in unregulated initiation of S phase. Tax inhibition of histone gene expression could allow incomplete chromatin assembly during DNA replication in S phase leading to increased incidence of DNA double strand breaks and greater chromosome instability. In normal somatic cells, histone gene expression is tightly coupled to DNA replication in S phase and is regulated exclusively at the mRNA level. We are now investigating whether Tax represses linker histone H1 and core histone gene expression through an effect on NPAT, SLBP or HIRA levels or activity, which function downstream of cyclin E-Cdk2 in histone gene regulation (Figure 4).

Significance

What mechanisms regulate gene expression? This is one of the most critical questions in biomedical science today. Many types of cancer result from mutations in the cellular machinery regulating gene expression. At the heart of this question is the mechanism and interactions of transcription regulation in the context of chromatin.

Graduate Mentor: Michael E. Dahmus, University of California, Davis. The functional significance of the C-terminal domain of RNA polymerase IIa/o.

Post-doctoral Mentor: James T. Kadonaga, University of California, San Diego. The role of core histones and the linker histone, H1, in transcriptional regulation using reconstituted chromatin templates.

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Moss, D. R. and Laybourn, P. J. Upstream Nucleosomes and Rgr1p Are Required for Nucleosomal Repression of Transcription, Mol. Micro. 36, 1-14 (2000).

Martens, C., Krett, B., and Laybourn, P. J. RNA Polymerase II and TBP Occupy the Repressed CYC1 Promoter, Mol. Micro. 40, 1009-1019 (2001).

Georges, S. A., Kraus, W. L., Luger, K., Nyborg, J. K., and Laybourn, P. J. p300-Mediated Tax Transactivation from Recombinant Chromatin: Histone Tail Deletion Mimics Coactivator Function, Mol. Cell Biol. 22, 127-137 (2002).

Livengood J. A., Scoggin K.E., Van Orden K., McBryant S. J., Edayathumangalam R. S., Laybourn P. J., Nyborg J. K. "p53 Transcriptional activity is mediated through the SRC1-interacting domain of CBP/p300," J. Biol. Chem. 277(11), 9054-9061 (2002).

Lemasson I., Polakowski N. J., Laybourn P. J., Nyborg J. K. "Transcription factor binding and histone modifications on the integrated proviral promoter in human T-cell leukemia virus-I-infected T-cells," J. Biol. Chem. 277(51), 49459-49465 (2002).

Marilley M., Radebaugh C. A., Geiss G. K., Laybourn P. J., Paule M. R. "DNA structural variation affects complex formation and promoter melting in ribosomal RNA transcription," Mol Genet Genomics. 267(6), 781-791 (2002).

Terrell, A. R., Wongwisansri, S., Pilon, J. L. and Laybourn, P. J. Reconstitution of Nucleosome Positioning, Remodeling, and Transcriptional Activation on the PHO5 Promoter, J. Biol. Chem. 277, 31038-31047 (2002).

Georges, S. A., Giebler, H. A., Cole, P. A., Luger, K., Laybourn, P. J., and Nyborg, J. K. "Tax Recruitment of CBP/p300, via the KIX Domain, Reveals a Potent Requirement for Acetyltransferase Activity That Is Chromatin Dependent and Histone Tail Independent." Mol. Cell Biol. 23, 3392-3404 (2003).

McBryant SJ, Abernathy SM, Laybourn PJ, Nyborg JK, Luger K.Preferential binding of the histone (H3-H4)2 tetramer by NAP1 is mediated by the amino terminal histone tails, J. Biol. Chem. 278, 44574-44583 (2003).

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Bao, Y., K. Konesky, Y. J. Park, S. Rosu, P.N. Dyer, D. Rangasamy, D. J. Tremethick, P.J. Laybourn, K. Luger. 2004, Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA, EMBO J. 23, 3314-3324.

Wongwisansri, S. and Laybourn, P. J. Disruption of the histone deacetylase gene RPD3 accelerates PHO5 activation kinetics through inappropriate Pho84p recycling, Euk. Cell 4, in press (2005).