Boosting student performance with inclusive writing-to-learn assignments through graphic organizers in large enrollment undergraduate biology courses.

Logistical challenges in large enrollment classes are often mentioned as obstacles to active learning. Writing is an integral part of being a scientist and is often one of the first tools considered by STEM instructors to increase student engagement, but iterative writing assignments in large classes require creativity on the part of the instructor. We found an association between writing-to-learn assignments designed to be consistent with inclusive learning pedagogies and student performance measures in a large enrollment undergraduate biology course. They provide ample opportunity for deliberate practice and inclusive engagement, components of the "heads and hearts" hypothesis posed to explain the variation in active learning impacts on the performance of minoritized students.

Human T Lymphotropic Virus Type 1 protein Tax reduces histone levels.

BACKGROUND: Human T-Lymphotropic Virus Type-1 (HTLV-1) is an oncogenic retrovirus that causes adult T-cell leukemia/lymphoma (ATLL). The virally encoded Tax protein is thought to be necessary and sufficient for T-cell leukemogenesis. Tax promotes inappropriate cellular proliferation, represses multiple DNA repair mechanisms, deregulates cell cycle checkpoints, and induces genomic instability. All of these Tax effects are thought to cooperate in the development of ATLL. RESULTS: In this study, we demonstrate that histone protein levels are reduced in HTLV-1 infected T-cell lines (HuT102, SLB-1 and C81) relative to uninfected T-cell lines (CEM, Jurkat and Molt4), while the relative amount of DNA per haploid complement is unaffected. In addition, we show that replication-dependent core and linker histone transcript levels are reduced in HTLV-1 infected T-cell lines. Furthermore, we show that Tax expression in Jurkat cells is sufficient for reduction of replication-dependent histone transcript levels. CONCLUSION: These results demonstrate that Tax disrupts the proper regulation of replication-dependent histone gene expression. Further, our findings suggest that HTLV-1 infection uncouples replication-dependent histone gene expression and DNA replication, allowing the depletion of histone proteins with cell division. Histone proteins are involved in the regulation of all metabolic processes involving DNA including transcription, replication, repair and recombination. This study provides a previously unidentified mechanism by which Tax may directly induce chromosomal instability and deregulate gene expression through reduced histone levels.

Biochemical analyses of transcriptional regulatory mechanisms in a chromatin context.

We have optimized a recombinant chromatin assembly system that properly incorporates core histones and histone H1 into a chromatin template containing a natural promoter sequence. This article provides a step-by-step procedure for expression and purification of the proteins required for assembling well-defined chromatin templates. We describe how to measure the degree of chromatin assembly in the absence and presence of histone H1 using topological analysis and how to perform micrococcal nuclease digestion to confirm H1 incorporation and determine the quality of in vitro chromatin templates. Further, we describe the use of sucrose gradient ultracentrifugation to verify that no unincorporated H1 remains as a second means for deciding on the proper H1 to core histone ratio during assembly. Additionally, we discuss the use of both yeast and Drosophila NAP-1 (yNAP-1 and dNAP-1, respectively) in the assembly of H1-containing chromatin. Finally, we provide detailed description of functional assays for investigating the mechanism of transcriptional regulation in a chromatin context (transcription, histone acetyltransferase activity, and protein association with promoter-bound complexes using immobilized chromatin templates).

Tax abolishes histone H1 repression of p300 acetyltransferase activity at the human T-cell leukemia virus type 1 promoter.

Upon infection of human T-cell leukemia virus type 1 (HTLV-1), the provirus is integrated into the host cell genome and subsequently packaged into chromatin that contains histone H1. Consequently, transcriptional activation of the virus requires overcoming the environment of chromatin and H1. To efficiently activate transcription, HTLV-1 requires the virally encoded protein Tax and cellular transcription factor CREB. Together Tax and CREB interact with three cis-acting promoter elements called viral cyclic-AMP response elements (vCREs). Binding of Tax and CREB to the vCREs promotes association of p300/CBP into the complex and leads to transcriptional activation. Therefore, to fully understand the mechanism of Tax transactivation, it is necessary to examine transcriptional activation from chromatin assembled with H1. Using a DNA template harboring the complete HTLV-1 promoter sequence and a highly defined recombinant assembly system, we demonstrate proper incorporation of histone H1 into chromatin. Addition of H1 to the chromatin template reduces HTLV-1 transcriptional activation through a novel mechanism. Specifically, H1 does not inhibit CREB or Tax binding to the vCREs or p300 recruitment to the promoter. Rather, H1 directly targets p300 acetyltransferase activity. Interestingly, in determining the mechanism of H1 repression, we have discovered a previously undefined function of Tax, overcoming the repressive effects of H1-chromatin. Tax specifically abrogates the H1 repression of p300 enzymatic activity in a manner independent of p300 recruitment and without displacement of H1 from the promoter.

Tax-dependent displacement of nucleosomes during transcriptional activation of human T-cell leukemia virus type 1.

The human T-cell leukemia virus type 1 (HTLV-1) is integrated into the host cell DNA and assembled into nucleosomes. Within the repressive chromatin environment, the virally encoded Tax protein mediates the recruitment of the coactivators CREB-binding protein/p300 to the HTLV-1 promoter, located within the long terminal repeats (LTRs) of the provirus. These proteins carry acetyltransferase activity that is essential for strong transcriptional activation of the virus in the context of chromatin. Consistent with this, the amino-terminal tails of nucleosomal histones at the viral promoter are acetylated in Tax-expressing cells. We have developed a system in which we transfect Tax into cells carrying integrated copies of the HTLV-1 LTR driving the luciferase gene to analyze changes in "activating" histone modifications at the LTR. Unexpectedly, Tax transactivation led to an apparent reduction of these modifications at the HTLV-1 promoter and downstream region that correlates with a similar reduction in histone H3 and linker histone H1. Micrococcal nuclease protection analysis showed that less LTR-luciferase DNA is nucleosomal in Tax-expressing cells. Furthermore, nucleosome depletion correlated with RNA polymerase II recruitment and loss of SWI/SNF. The M47 Tax mutant, deficient in HTLV-1 transcriptional activation, was also defective for nucleosome depletion. Although this mutant formed complexes with CREB and p300 at the HTLV-1 promoter in vivo, it was unable to mediate RNA polymerase II recruitment or SWI/SNF displacement. These results support a model in which nucleosomes are depleted from the LTR and transcribed region during Tax-mediated transcriptional activation and correlate RNA polymerase II recruitment with nucleosome depletion.

Disruption of histone deacetylase gene RPD3 accelerates PHO5 activation kinetics through inappropriate Pho84p recycling.

The histone deacetylase Rpd3p functions as a transcriptional repressor of a diverse set of genes, including PHO5. Here we describe a novel role for RPD3 in the regulation of phosphate transporter Pho84p retention in the cytoplasmic membrane. We show that under repressing conditions (with P(i)), PHO5 expression is increased in a pho4Delta rpd3Delta 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 (without P(i)), PHO5 transcripts accumulated more rapidly in rpd3Delta cells. This more rapid response correlates with a defect in phosphate uptake due to premature recycling of Pho84p, the high-affinity H+/PO4(3-) symporter. 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.

Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA.

H2A.Bbd is an unusual histone variant whose sequence is only 48% conserved compared to major H2A. The major sequence differences are in the docking domain that tethers the H2A-H2B dimer to the (H3-H4)(2) tetramer; in addition, the C-terminal tail is absent in H2A.Bbd. We assembled nucleosomes in which H2A is replaced by H2A.Bbd (Bbd-NCP), and found that Bbd-NCP had a more relaxed structure in which only 118+/-2 bp of DNA is protected against digestion with micrococcal nuclease. The absence of fluorescence resonance energy transfer between the ends of the DNA in Bbd-NCP indicates that the distance between the DNA ends is increased significantly. The Bbd docking domain is largely responsible for this behavior, as shown by domain-swap experiments. Bbd-containing nucleosomal arrays repress transcription from a natural promoter, and this repression can be alleviated by transcriptional activators Tax and CREB. The structural properties of Bbd-NCP described here have important implications for the in vivo function of this histone variant and are consistent with its proposed role in transcriptionally active chromatin.

Transcription regulatory complexes bind the human T-cell leukemia virus 5' and 3' long terminal repeats to control gene expression.

The human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that integrates randomly into the T-cell genome. Two long terminal repeats (LTRs) flank the integrated provirus. The upstream and downstream LTRs carry identical promoter sequences. Studies with other retroviruses suggest that the downstream promoter is silent and that RNA polymerases initiating at the upstream promoter proceed through the 3' LTR. In this study, we used the chromatin immunoprecipitation assay to compare the binding of transcription regulatory proteins at both the upstream and downstream promoters in HTLV-1-infected cell lines and adult T-cell leukemia-lymphoma cells. Unexpectedly, we detected a nearly equal distribution of activator (Tax, CREB, ATF-1, ATF-2, c-Fos, and c-Jun) and regulatory protein (CBP, p300, TAF(II)250, and polymerase II) binding at both the upstream and downstream promoters. Consistent with this observation, we found that the downstream promoter was transcriptionally active, suggesting that the two promoters are functionally equivalent. We also detected asymmetrical binding of histone deacetylases (HDAC-1, -2, and -3) at both promoters. All three HDACs strongly repressed Tax transactivation, and this repression correlated with displacement of Tax from the HTLV-1 promoter. These effects were reciprocal, as Tax expression reversed HDAC repression and displaced HDACs from the HTLV-1 promoter. These data suggest that HTLV-1 transcriptional regulation at both the 5' and 3' LTRs is mediated, in part, through the mutually exclusive binding of Tax and HDACs at the proviral promoters.

Preferential binding of the histone (H3-H4)2 tetramer by NAP1 is mediated by the amino-terminal histone tails.

The yeast nucleosome assembly protein 1 (yNAP1) participates in many diverse activities, such as the assembly of newly synthesized DNA into chromatin and the rearrangement of nucleosomes during transcriptional activation. yNAP1 does not require ATP hydrolysis to perform these functions and is a valuable tool for in vitro chromatin assembly. Using recombinant histone complexes, we show that yNAP1 has a preference for binding the (H3-H4)2 tetramer over the (H2A-H2B) dimer. We find that the loss of the histone tails abrogates this preference for H3 and H4, and we demonstrate a direct interaction between yNAP1 and the amino-terminal tails of H3 and H4. yNAP1 binds to one histone fold domain, thus specifying the stoichiometry of the complexes formed with the histone dimer and tetramer. Finally, we provide evidence that the acidic carboxyl-terminal region of yNAP1, although dispensable for nucleosome assembly in vitro, contributes to binding via structure-independent electrostatic interactions. Our results are consistent with recent mechanistic investigations of NAP1 and expand our understanding of the histone chaperone family of assembly factors.

Tax recruitment of CBP/p300, via the KIX domain, reveals a potent requirement for acetyltransferase activity that is chromatin dependent and histone tail independent.

Robust transcription of human T-cell leukemia virus type 1 (HTLV-1) genome requires the viral transactivator Tax. Although Tax has been previously shown to interact with the KIX domain of CBP/p300 in vitro, the precise functional relevance of this interaction remains unclear. Using two distinct approaches to interrupt the physical interaction between Tax and KIX, we find that Tax transactivation from chromatin templates is strongly dependent on CBP/p300 recruitment via the KIX domain. Additionally, we find that the primary functional contribution of CBP/p300 to Tax transactivation resides in the intrinsic acetyltransferase activity of the coactivators. These studies unexpectedly uncover a specific requirement for CBP/p300 acetyltransferase activity on chromatin templates assembled with nucleosomes lacking their amino-terminal tails. Together, these data indicate that the KIX domain of CBP/p300 is essential for targeting the acetyltransferase activity of the coactivator to the Tax-CREB (Tax/CREB) complex. Significantly, these observations reveal the presence of one or more CBP/p300 acetyltransferase targets that function specifically on chromatin templates, are independent of the histone tails, and are critical to Tax transactivation.

Transcription factor binding and histone modifications on the integrated proviral promoter in human T-cell leukemia virus-I-infected T-cells.

The human T-cell leukemia virus (HTLV-I)-encoded Tax protein is a potent transcriptional activator that stimulates expression of the integrated provirus. Biochemical studies indicate that Tax, together with cellular transcription factors, interacts with viral cAMP-response element enhancer elements to recruit the pleiotropic coactivators CREB-binding protein and p300. Histone acetylation by these coactivators has been shown to play a major role in activating HTLV-I transcription from chromatin templates in vitro. However, the extent of histone modification and the precise identity of the cellular regulatory proteins bound at the HTLV-I promoter in vivo is not known. Chromatin immunoprecipitation analysis was used to investigate factor binding and histone modification at the integrated HTLV-I provirus in infected T-cells (SLB-1). These studies reveal the presence of Tax, a variety of ATF/CREB and AP-1 family members (CREB, CREB-2, ATF-1, ATF-2, c-Fos, and c-Jun), and both p300 and CREB-binding protein at the HTLV-I promoter. Consistent with the binding of these coactivators, we observed histone H3 and H4 acetylation at three regions within the proviral genome. Histone deacetylases were also present at the viral promoter and, following their inhibition, we observe an increase in histone H4 acetylation on the HTLV-I promoter and a concomitant increase in viral RNA. Together, these results suggest that a variety of transcriptional activators, coactivators, and histone deacetylases participate in the regulation of HTLV-I transcription in infected T-cells.

Reconstitution of nucleosome positioning, remodeling, histone acetylation, and transcriptional activation on the PHO5 promoter.

The PHO5 gene promoter is an important model for the study of gene regulation in the context of chromatin. Upon PHO5 activation the chromatin structure is reconfigured, but the mechanism of this transition remains unclear. Using templates reconstituted into chromatin with purified recombinant yeast core histones, we have investigated the mechanism of chromatin structure reconfiguration on the PHO5 promoter, a prerequisite for transcriptional activation. Footprinting analyses show that intrinsic properties of the promoter DNA are sufficient for translational nucleosome positioning, 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 histone-modifying or nucleosome-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. The addition of acetyl-CoA results in significant core histone acetylation. These findings indicate that transcriptional activation requires Pho4p, Pho2p, nucleosome remodeling, and nucleosome acetylation. Furthermore, we find that DNA binding, nucleosome remodeling, and transcriptional activation are separable steps, facilitating biochemical analysis of the PHO5 regulatory mechanism.

p53 Transcriptional activity is mediated through the SRC1-interacting domain of CBP/p300.

The tumor suppressor p53 recruits the cellular coactivator CBP/p300 to mediate the transcriptional activation of target genes. In this study, we identify a novel p53-interacting region in CBP/p300, which we call CR2, located near the carboxyl terminus. The 95-amino acid CR2 region (amino acids 2055--2150) is located adjacent to the C/H3 domain and corresponds precisely with the minimal steroid receptor coactivator 1 (SRC1)-interacting domain of CBP (also called IBiD). We show that the region of p53 that participates in the CR2 interaction resides within the first 107 amino acids of the protein. p53 binds strongly to the CR2 domain of both CBP and the highly homologous coactivator p300. Importantly, an in-frame deletion of CR2 within the full-length p300 protein strongly compromises p300-mediated p53 transcriptional activation from a chromatin template in vitro. The identification of the p53-interacting CR2 domain in CBP/p300 prompted us to ask if the human T-cell leukemia virus (HTLV-I) Tax protein, which also interacts with CR2, competes with p53 for binding to this domain. We show that p53 and Tax exhibit mutually exclusive binding to the CR2 region, possibly contributing to the previously reported Tax repression of p53 function. Together, these studies identify and molecularly characterize a new p53 binding site on CBP/p300 that participates in coactivator-mediated p53 transcription function. The identity of the p53.CR2 interaction indicates that at least three distinct sites on CBP/p300 may participate in mediating p53 transactivation.

p300-mediated tax transactivation from recombinant chromatin: histone tail deletion mimics coactivator function.

Efficient transcription of the human T-cell leukemia virus type 1 (HTLV-1) genome requires Tax, a virally encoded oncogenic transcription factor, in complex with the cellular transcription factor CREB and the coactivators p300/CBP. 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 HTLV-1 promoter assembled into chromatin activated transcription several hundredfold. Chromatin templates selectively lacking amino-terminal histone tails demonstrated enhanced transcriptional activation by Tax and CREB, with significantly reduced dependence on p300 and acetyl coenzyme A (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 the major targets of p300 and that tail deletion and acetylation are functionally equivalent.