A key role for chd1 in histone h3 dynamics at the 3\u27 ends of long genes in yeast

Chd proteins are ATP-dependent chromatin remodeling enzymes implicated in biological functions from transcriptional elongation to control of pluripotency. Previous studies of the Chd1 subclass of these proteins have implicated them in diverse roles in gene expression including functions during initiation, elongation, and termination. Furthermore, some evidence has suggested a role for Chd1 in replication-independent histone exchange or assembly. Here, we examine roles of Chd1 in replication-independent dynamics of histone H3 in both Drosophila and yeast. We find evidence of a role for Chd1 in H3 dynamics in both organisms. Using genome-wide ChIP-on-chip analysis, we find that Chd1 influences histone turnover at the 5\u27 and 3\u27 ends of genes, accelerating H3 replacement at the 5\u27 ends of genes while protecting the 3\u27 ends of genes from excessive H3 turnover. Although consistent with a direct role for Chd1 in exchange, these results may indicate that Chd1 stabilizes nucleosomes perturbed by transcription. Curiously, we observe a strong effect of gene length on Chd1\u27s effects on H3 turnover. Finally, we show that Chd1 also affects histone modification patterns over genes, likely as a consequence of its effects on histone replacement. Taken together, our results emphasize a role for Chd1 in histone replacement in both budding yeast and Drosophila melanogaster, and surprisingly they show that the major effects of Chd1 on turnover occur at the 3\u27 ends of genes

ISEE's Framework of Six Elements to Guide the Design, Teaching, and Assessment of Authentic and Inclusive STEM Learning Experiences

It seems intuitive that effective learning experiences in science, technology, engineering and mathematics (STEM) should be inclusive and should mirror authentic STEM as practiced by professionals. However, it is less intuitive what an authentic, inclusive STEM learning experience (AISLE) should look like or include. Over the course of 20 years, the Institute for Scientist & Engineer Educators (ISEE) has grappled with this question, developing and refining a framework of six key elements of authentic and inclusive STEM learning experiences. Here, we present this framework, which grew from an exploration of what “scientific inquiry” means in the context of teaching and learning, and expanded to include practices and norms that are valued in engineering fields. ISEE’s framework is the cornerstone of its Professional Development Program (PDP), which trained early-career science and engineering professionals to teach STEM effectively, primarily at the college level, from 2001-2020. In addition to presenting the six elements of this framework, we describe how PDP participants implemented the elements, and we provide recommendations for putting the elements into practice through the design, teaching and assessment of STEM learning experiences

Recommendations from 20 Years of Professional Development of Early-Career Scientists and Engineers

The Professional Development Program (PDP) was a highly impactful and innovative program that was run by the Institute for Scientist & Engineer Educators for twenty years, from 2001–2020. The program trained early-career scientists and engineers to teach effectively and inclusively, while also developing participants’ skills in leadership, collaboration, and teamwork. In this paper, we summarize important aspects of the PDP and some of the program’s major outcomes, describe legacies of the program, and share recommendations based on two decades of experience. A large section of this paper details aspects of the PDP that we consider essential to the program but that might not be apparent from other documentation of the program. Recommendations for others interested in professional development of STEM graduate students and postdoctoral scholars are: 1) invest in establishing program culture; 2) prepare participants pursuing all STEM career paths for inclusive teaching; 3) focus on teaching and learning authentic STEM practices of participants’ fields; 4) provide authentic and challenging contexts for practicing professional skills; 5) model all aspects of what participants are expected to do; and 6) provide opportunities for growth and becoming a collaborator within the community

Length dependence of Chd1 effects on H3 replacement.

<p>H3 replacement was averaged for the 500 bp at the 5′ ends of genes (A), or the 3′ ends of genes (B). Genes were ordered by length, and an 80 gene window average is shown for wild type and <i>chd1Δ</i> turnover data as indicated. Bottom panel plots gene lengths, and locations for 1, 2, and 3 kb are indicated below panel (B).</p

The H3 N-terminal tail functions redundantly with Chd1 and an H3.3-like surface of histone H3 in budding yeast.

<p>The indicated histone H3 plasmids (which also carried histone H4) were transformed into wild type <i>CHD1</i> or <i>chd1</i> null strains that lack both chromosomal copies of the histone H3/H4 genes and contained a <i>URA3 H3/H4</i> plasmid. Cultures were adjusted to 1×10⁷ cells per ml and five-fold serial dilutions were spotted directly onto 5FOA media, selecting for cells that had lost the <i>URA3 H3/H4</i> plasmid, and incubated for 2 days at 30°C.</p

Chd1 effects on H3 methylation patterns.

<p>H3K4me3 and H3K36me3 were mapped genome-wide by ChIP-chip on tiling microarrays. Metagene analysis is shown for wild type and <i>chd1Δ</i> strains, as indicated.</p

Chd1 affects H3.3_core-GFP localization on Chd1 in <i>Drosophila</i>.

<p>(A) Representative sections from confocal imaging of H3.3_core-GFP in nuclei from salivary glands of wild type larvae, (B) <i>chd1⁵</i> heterozygotes and (C) <i>chd1⁵</i> homozygotes. The GFP signal is pseudo green. In all cases, H3.3_core-GFP was expressed from <i>P[UHS-H3.3_core-GFP]</i> and driven by <i>P{GawB} AB1-Gal4</i>. (D) Quantitation of banding patterns observed in nuclei from flies with the indicated genotypes. A total of 44 wild type, 144 heterozygote, and 162 homozygous null nuclei were scored, all blind to genotype.</p

Confirmation of the cellular targets of benomyl and rapamycin using next-generation sequencing of resistant mutants in S. cerevisiae

Investigating the mechanisms of action (MOAs) of bioactive compounds and the deconvolution of their cellular targets is an important and challenging undertaking. Drug resistance in model organisms such as S. cerevisiae has long been a means for discovering drug targets and MOAs. Strains are selected for resistance to a drug of interest, and the resistance mutations can often be mapped to the drug’s molecular target using classical genetic techniques. Here we demonstrate the use of next generation sequencing (NGS) to identify mutations that confer resistance to two well-characterized drugs, benomyl and rapamycin. Applying NGS to pools of drug-resistant mutants, we develop a simple system for ranking single nucleotide polymorphisms (SNPs) based on their prevalence in the pool, and for ranking genes based on the number of SNPs that they contain. We clearly identified the known targets of benomyl (TUB2) and rapamycin (FPR1) as the highest-ranking genes under this system. The highest-ranking SNPs corresponded to specific amino acid changes that are known to confer resistance to these drugs. We also found that by screening in a pdr1Δ null background strain that lacks a transcription factor regulating the expression of drug efflux pumps, and by pre-screening mutants in a panel of unrelated anti-fungal agents, we were able to mitigate against the selection of multi-drug resistance (MDR) mutants. We call our approach “Mutagenesis to Uncover Targets by deep Sequencing, or “MUTseq”, and show through this proof-of-concept study its potential utility in characterizing MOAs and targets of novel compounds.(